System and method for extracting a target moiety from a sample using acoustic droplet ejection

ABSTRACT

A method and system are provided for extracting a target analyte from a sample using acoustic ejection technology. The method involves applying focused acoustic energy to a fluid reservoir housing a fluid composition that contains a target analyte and comprises an upper region and a lower region, where the concentration of the target analyte in the upper region differs from that in the lower region. The focused acoustic energy is applied in a manner that is effective to result in the ejection of a fluid droplet from from the fluid composition into a droplet receiver, wherein the concentration of the analyte in the droplet corresponds to either the concentration of the analyte in the upper region or the concentration of the analyte in the lower region, and wherein the concentration of the analyte is substantially uniform throughout the droplet. The fluid composition may comprise an ionic liquid, used in the extraction of ionic target analytes. Related methods and an acoustic extraction system are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. Ser. No. 17/108,931, filed Dec. 1, 2020,which is a continuation of U.S. Ser. No. 16/292,177, filed Mar. 4, 2019,which claims priority under 35 U.S.C. § 119(e)(1) to provisional U.S.Patent Application Ser. No. 62/638,143, filed Mar. 3, 2018, thedisclosures of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION (1) Technical Field

The present invention relates generally to systems and methods forextracting a target analyte from a fluid composition using acousticdroplet ejection. The invention finds utility in numerous fields,including chemistry and biology.

(2) Description of Related Art

The extraction of molecules from a mixture is an essential step inprocesses employed in numerous technical areas, including syntheticorganic chemistry, materials science, pharmaceutical research anddevelopment, and molecular biology. Although now required in manycontexts, the extraction of biomolecules such as nucleic acids andproteins is particularly challenging insofar as as they are normallycontained within complex host environments such as cells, tissues, andblood. Efficient and effective extraction of DNA, RNA, and proteins isnevertheless necessary in numerous processes and products. Diagnostickits, identity and relationship testing, pathogen detection, tissuetyping, and genetic research are just several examples.

Purification of DNA can involve the separation and removal ofchromosomal and/or mitochondrial DNA from a biological environment, orit may be carried out in the context of isolating recombinant DNAconstructs, i.e., DNA containing a recombinant sequence, such as aplasmid. Polymerase chain reaction (PCR) amplification of DNA,diagnostic testing procedures, and a host of sensitive assays require ahigh degree of purity. Contaminants in a DNA sample can inhibit one ormore critical steps of a diagnostic or analytical procedure. Somecontaminants, for example, can inhibit the polymerase chain reaction orthe action of restriction enzymes.

Any DNA purification method requires (1) effective disruption of abiological host environment, e.g., cells or tissues, that contains thetarget DNA; (2) denaturation of proteins and nucleoprotein complexesusing a protease and/or denaturant; (3) inactivation of endogenousnucleases; and (4) removal of the target DNA from the sample. Theisolated target DNA should be free of any compounds or materialsoriginally present, e.g., proteins, lipids, RNA, other nucleic acids,and the like, and in most cases the purification process must avoid DNAfragmentation resulting from mechanical shearing or the presence ofcontaminants. Isolation of RNA is even more complicated, insofar as RNAis inherently unstable, strong denaturants are necessary to inhibitendogenous RNAses, RNAses are heat-stable and refold following thermaldenaturation, and the RNAses, lacking cofactors, are difficult toinactivate.

In nucleic acid purification, disruption of cells or tissues is usuallycarried out using a detergent to disrupt the lipid bilayer of the cellmembrane. Detergents disrupt both lipid-lipid and lipid-proteininteractions in the cell membrane, enabling solubilization of membranecomponents. With organisms that contain cell walls in addition to a cellmembrane, additional treatment may be required; for instance, treatmentwith lysozyme is necessary to digest the peptidoglycan cell wall ofgram-positive bacteria and treatment with lyticase or zymolase isrequired to disrupt the polysaccharide cell wall of yeasts.

Denaturation of proteins and nucleoproteins involves modification ofprotein conformation by disruption of secondary structure, and iscarried out using protein denaturing agents such as ionic detergents,chaotropic agents, reducing agents, heat, and/or proteases. In mammaliancells, DNA is compacted with histones in a macromolecular nucleoproteinstructure (i.e., chromatin), and denaturation enables release ofchromosomal DNA from the nucleoprotein complex. Chelating agents such asethylenediaminetetraacetic acid (EDTA) are typically used to inactivatenucleases, as is Proteinase K. Some commercial systems require stepwiseprocessing, i.e., cell lysis, denaturation, and nuclease inactivation,and other systems provide a single solution containing components forcarrying out all three of the aforementioned steps.

Ultimately, the target DNA must be isolated from the treated biologicalsample, which will likely contain proteins, protein fragments, lipids,carbohydrates, salts, and cell debris. Historically, DNA was purifiedvia liquid-liquid extraction. The aqueous cell hydrolysate was shakenwith a phenol-chloroform mixture, optionally containing some isoamylalcohol to inhibit RNAse activity. This mixture separates into twolayers with the hydrophobic chloroform-phenol lower phase containing theproteins, lipids, carbohydrates, and cell debris, while nucleic acidsremain in the upper aqueous phase. The aqueous DNA solution of the upperphase is collected, the DNA is precipitated from the supernatant, andthe DNA precipitate is rinsed and dissolved with buffer. The techniqueis cumbersome and time-consuming, however, and, as has been widelynoted, chloroform is highly toxic and phenol is flammable, corrosive,and toxic as well.

Liquid-liquid extraction has been largely replaced with a solid-phasenucleic acid purification method using centrifuge-based columns. In thiscase, the cell lysate is mixed with a buffer solution and either achaotropic agent or a short-chain alcohol. The lysate is transferred toa column and centrifuged to drive the liquid through the solid phase,which has been surface treated to retain the negatively charged nucleicacid. Proteins and other contaminants are washed through the columnwhile nucleic acids bind to it. After washing steps, the nucleic acidsare eluted with water or buffer (e.g., dilute TRIS-EDTA buffer at a pHof about 8.4). Mixed-bed solid phase extraction has also been disclosed;see U.S. Pat. No. 6,376,194 to Smith et al. While easier and safer thanconventional liquid-liquid extraction employing the phenol-chloroformtechnique, column-based DNA extraction still requires manualintervention and is not easily automated.

In a variation on traditional solid phase separation, a magneticbead-based nucleic acid purification method has been developed. In thisprocess, magnetic beads having coated or otherwise treated surfaces bindnucleic acids in the presence of a chaotropic agent. The beads arecombined with a biological sample, and nucleic acid in the sample bindsto the bead surface. A magnet is used to pull the beads withsurface-bound nucleic acid to a stable position in a microplate well,centrifuge tube, or other vessel. Once the beads are magneticallyimmobilized, the supernatant containing the impurities is removed, thebeads are washed with clean wash buffer, and the nucleic acids aredisplaced from the magnetic beads with a small volume of dilute elutionbuffer. By eliminating centrifugation, the technique is generally moreamenable to automation and higher throughput than other solid phasepurification methods. However, the cost to clean up each sample issubstantial

Various types of extraction techniques for isolating and purifying DNA,RNA, and proteins are described in Tan et al. (2009) J. Biomed.Biotech., Article ID 574398. As explained therein, there is an ongoingneed for improved ways to isolate and purify biomolecules. Tan et al.note that automation of extraction procedures is desirable in order toreduce working time, decrease labor costs, enhance worker safety, and,ideally, increase both the reproducibility and the quality of results.Tan et al. further note that commercially available automated systemsare somewhat limited insofar as they tend to be large, expensive, andcomplex, intended for use in medium to large laboratories, while morerecent automation processes adapted for small and medium samplethroughput still require a time-consuming extraction process, on theorder of 20 to 40 minutes of processing time per sample. Proper liquidhandling is essential, both for each extraction step of an automatedprocedure and for transferring liquids as necessary; optimally, asexplained by Tan et al., robotic workstations should be fully automatedand thereby obviate the need for pre-processing steps. Tan et al.additionally point out that continued improvement in miniaturization isnecessary, and would remedy a weakness of available extraction systems.

Acoustic droplet ejection (ADE) is a methodology that has been disclosedas useful in the ejection of immiscible fluids; see U.S. Pat. Nos.6,548,308 and 6,642,061 to Ellson et al. The aforementioned patentsdescribe the use of ADE to eject droplets from immiscible liquids onto asubstrate surface, where the droplets generally have a first regioncorresponding to one of the liquids and a second region corresponding tothe other liquid. ADE has not been implemented in the extraction oftarget moieties from a sample, however, and it is well known thatdevelopment of extraction techniques can be complicated and problematic,as explained by Tan et al., supra.

An ideal extraction system and method would accomplish at least thefollowing goals:

Provide the isolated target molecule in high purity;

Be capable of use to extract any of a wide variety of target molecules;

Yield accurate, consistent, and reproducible results;

Be fully automated;

Be capable of use under standard laboratory conditions without need forhigh temperatures or an inert atmosphere;

Minimize per-sample processing time and enable high-throughput sampleprocessing;

Allow effective and efficient processing of very small sample sizes, onthe order of nanoliters or smaller; accuracy and reliability;

Eliminate the need for toxic, volatile solvents;

Rely on reagents that can be recycled and reused in a subsequentextraction step; and

Enable rapid introduction of the extracted target moiety into analyticaldevice such as a mass spectrometer.

SUMMARY OF THE INVENTION

The invention is addressed to the above-mentioned needs in the art andprovides a method and system for extracting a target analyte usingacoustic droplet ejection (ADE) technology. The target analyte is in afluid composition, e.g., dissolved in a solvent or solvent mixture, andmay comprise a single analyte or a mixture of analytes in amulti-component composition. The fluid composition may comprise abiological sample such as living tissue, cells, blood, or the like,which may or may not have been processed in some manner prior toextraction. The invention employs ADE technology and, in a preferredembodiment, liquid-liquid partitioning, making use of liquids that, forexample, have different affinities for various types of components in asample, and/or liquids in which the solubility of a component ofinterest is different. The method and system of the invention can bereadily implemented in the high-throughput context and are useful inextracting a variety of target molecules from samples, including smallvolumes of biological samples composed of complex biological mixtures.

The present extraction method encompasses the partial or completeremoval of a target analyte from an initial fluid composition, and alsoencompasses a separation process in which a final fluid compositioncontains a non-target component at a lower concentration than in theinitial fluid composition.

In a first embodiment of the invention, a method is provided forproducing a fluid droplet containing a target analyte at a selectedconcentration. The method comprises: (a) providing, in a fluidreservoir, a fluid composition that contains the target analyte andcomprises an upper region and a lower region, wherein the analyte is ata first concentration in the upper region and at a second concentrationin the lower region, and further wherein the second concentration isdifferent from the first concentration; and (b) applying focusedacoustic energy to the fluid reservoir in a manner effective to eject afluid droplet from the fluid composition into a droplet receiver,wherein the ejected droplet comprises the target analyte at the selectedconcentration, and further wherein the selected concentration is (i)substantially equivalent to either the first concentration or the secondconcentration, and (ii) substantially uniform throughout the droplet.The fluid composition in the fluid reservoir generally comprises asample that contains the target analyte, e.g., a biological sample. Thebiological sample may comprise a sample dissolved or suspended in afluid or the biological sample may itself be fluidic.

In one aspect of the aforementioned embodiment, the upper region of thefluid composition comprises an upper layer of a first liquid, while thelower region of the fluid composition comprises a lower layer of asecond liquid. Depending on the target analyte, the first liquid, andthe second liquid, the analyte may partition into the first liquid orthe second liquid preferentially. That is, the first and second liquidsare selected such that the first liquid has a first affinity for thetarget analyte and the second liquid has a second affinity for thetarget analyte, and the first affinity and the second affinity aredifferent. For example, with a hydrophilic, e.g., ionic, target analyte,a hydrophilic upper liquid, and a hydrophobic lower liquid, the targetanalyte will tend to partition into the upper, hydrophilic liquid. Asanother example, when the solubility of the target analyte is greater inthe lower liquid than in the upper liquid, the target analyte will tendto partition into the lower liquid. The first liquid and the secondliquid may differ in volatility, density, viscosity, and/or otherphysical or chemical characteristics.

In a related aspect, the solubility of the target analyte in the lowerliquid differs from its solubility in the upper liquid by at least about50%.

In another related aspect, the solubility of the target analyte in thelower liquid differs from its solubility in the upper liquid by at leastabout 85%.

In another aspect of the aforementioned embodiment, the target analyteis an ionic target analyte, i.e., an analyte that is ionized at aselected pH, e.g., a pH in the range of about 6 to about 8. An ionictarget analyte may be a negatively charged moiety or a positivelycharged moiety, in association with a cationic or anionic counterion,respectively.

In another aspect, the target analyte comprises a biomolecule. Thebiomolecule may be a nucleic acid, a peptide or protein, a lipidicmoiety, or the like. In a related aspect, the biomolecule comprises DNA.Peptides, proteins, and the like may have a molecular weight in therange of about 100 daltons to about 200 kilodaltons. Much larger targetanalytes are envisioned, however, insofar as the present invention isuseful in conjunction with large nucleic acid fragments, unfragmentedsingle-stranded or double-stranded DNA, an entire genome or more thanone entire genome, and intact cells.

In a further aspect, the fluid composition comprises an ionic liquid,i.e., a salt that is in the form of a liquid at the conditions used forextraction.

In a related aspect, the method employs an ionic liquid in the acousticejection of a charged biomolecule, such as a nucleic acid (e.g., DNA)from a fluid composition.

In another related aspect, the ionic liquid serves as a first liquid,and an aqueous liquid serves as a second liquid, where the aqueousliquid is buffered to a pH that alters the relative affinity of theionic analyte for the ionic liquid and the aqueous liquid.

In another aspect of the aforementioned embodiment, the method furtherincludes, prior to step (a): subjecting a combination of the sample anda miscible mixture of the first liquid and the second liquid to acondition that renders the two liquids substantially immiscible,resulting in the partitioning of the fluid composition into the upperregion and the lower region.

In another aspect of the embodiment, the droplet receiver comprises ananalytical instrument. In a related aspect, the analytical instrument isa mass spectrometer.

In another aspect of the embodiment, step (b) of the method is repeatedmultiple times to eject multiple fluid droplets into the dropletreceiver.

In another aspect of the embodiment, the droplet receiver comprises adroplet receiving reservoir. In a related aspect, step (b) of the methodis repeated multiple times until at least 20 wt. % of the target analyteis transferred from the fluid reservoir to the droplet receivingreservoir.

In a further aspect of the embodiment, the fluid reservoir is one of aplurality of reservoirs each housing a fluid composition containing atarget analyte, wherein any two of the fluid compositions may be thesame or different, and/or any two of the target analytes may be the sameor different. The plurality of reservoirs may be arranged in an arrayand/or contained within a substrate that comprises an integratedmultiple reservoir unit. In a related aspect, fluid droplets areacoustically ejected from an array of fluid reservoirs into acorresponding array of droplet receiving reservoirs.

In another aspect of the embodiment, the fluid composition in the fluidreservoir has a volume of no more than about 125 μL.

In another aspect of the embodiment, the ejected fluid droplet has avolume of no more than about 60 nL.

In another aspect of the embodiment, the fluid droplet has a volume ofno more than about 30 nL.

In a related aspect of this embodiment, acoustic droplet ejection iscarried out with respect to a plurality of fluid reservoirs insuccession, with rapid reservoir-to-reservoir transitions, e.g., at mostabout 0.5 seconds, or at most about 0.1 seconds, or at most about 0.001seconds.

In a further aspect of the embodiment, the interior surfaces of thefluid reservoir are coated with a surface coating composition. In arelated aspect, the surface coating is selected to repel or attract theupper fluid layer, thereby altering the shape of the meniscus and thethickness of the central region of the upper fluid layer.

In another aspect of the embodiment, the method further includesdetecting the presence of a liquid-liquid boundary between the upper andlower fluid layers. In a related aspect, fluid droplets are repeatedlyejected from the upper fluid layer until no liquid-liquid boundary isdetected, meaning that substantially all of the upper layer has beenremoved from the fluid composition and the acoustic ejection process canbe stopped.

In another embodiment of the invention, a method is provided forextracting an ionic target analyte from a sample, where the methodcomprises admixing the sample with an ionic liquid and a non-ionicliquid under conditions that facilitate partitioning of the ionicanalyte into the ionic liquid, and acoustically removing the non-ionicliquid from the mixture.

In an additional embodiment of the invention, a method is provided forextracting an ionic target analyte from a sample, where the methodcomprises admixing the sample with an ionic liquid and a non-ionicliquid under conditions that facilitate partitioning of the ionicanalyte into the ionic liquid to provide a solution of the ionic analytein the ionic liquid, removing the non-ionic liquid from the mixture, andacoustically ejecting droplets of the ionic analyte solution into adroplet receiver.

In another embodiment, the invention provides an extraction method thatcomprises: (a) providing, in a fluid reservoir, an initial fluidcomposition that contains the target analyte and comprises an upperlayer of a first liquid and a lower layer of a second liquid, whereinthe analyte is at a first concentration in the upper layer and at asecond concentration in the lower layer, wherein the secondconcentration is higher than the first concentration; and (b) repeatedlyapplying focused acoustic energy to the fluid reservoir in a mannereffective to eject fluid droplets of the upper layer of the fluid,thereby removing at least a portion of the upper layer while allowingthe lower layer to remain in the fluid reservoir.

In a further embodiment of the invention, a method is provided forextracting an ionic analyte from a biological sample, comprising: (a)acoustically ejecting droplets of a biological sample comprising theionic analyte and an aqueous medium into an ionic liquid contained in adroplet receiving reservoir; (b) inverting the droplet receivingreservoir, whereby an upper aqueous layer and a lower ionic liquid layercomprising ionic analyte are formed; and (c) removing the upper aqueouslayer to provide an ionic analyte solution comprising the ionic analytein the ionic liquid. The biological sample may be a processed biologicalsample, e.g., a sample containing lysed cells.

In one aspect of the embodiment, the aqueous medium comprises a buffersystem that maintains the biological sample at a first pH, wherein thefirst pH is selected so that at least 60 wt. % of the ionic analyte inthe biological sample partitions into the ionic liquid upon admixturetherewith.

In another aspect of the embodiment, the method further comprises, afterstep (c), step (d): admixing the ionic analyte solution with anextraction buffer having a second pH selected so that at least 60 wt. %of the ionic analyte in the ionic liquid partitions into the extractionbuffer.

In an additional embodiment, the invention provides a method foracoustically extracting DNA from an aqueous biological sample, where themethod comprises:

(a) admixing the aqueous biological sample with an ionic liquid in afluid reservoir under conditions effective to provide a fluidcomposition that comprises an upper aqueous layer and a lower ionicliquid layer;

(b) treating the fluid composition so that DNA in the biological samplepartitions into the lower ionic liquid layer;

(c) removing the upper aqueous layer so that a DNA solution in the ionicliquid remains in the fluid container;

(d) admixing the DNA solution with an extraction buffer having a pHselected so that at least 60 wt. % of the DNA in the ionic liquidpartitions into the extraction buffer; and

(e) successively acoustically ejecting droplets of the DNA-containingextraction buffer into a droplet receiver.

In another embodiment, the invention provides a method for extractinglipidic components from an aqueous biological sample, comprising:admixing the aqueous biological sample with an organic solvent in afluid reservoir, thereby providing a partitioned fluid composition withan upper organic layer comprising a lipid solution and a lower aqueouslayer; and successively acoustically ejecting droplets of the lipidsolution into a droplet receiver.

In a further embodiment of the invention, an acoustic extraction systemis provided for extracting an ionic target analyte from a sample,comprising: (a) a fluid reservoir housing a fluid composition comprisingthe ionic target analyte and an ionic liquid; and (b) an acousticdroplet ejector in acoustic coupling relationship with the fluidreservoir for generating acoustic radiation in a manner effective toeject a fluid droplet from the fluid composition into a dropletreceiver, the ejector comprising an acoustic radiation generator and afocusing means for focusing the acoustic radiation at a focal pointwithin the reservoir.

In one aspect of this embodiment, the system further includes thedroplet receiver, e.g., an analytical instrument such as a massspectrometer or the like, or a droplet receiving reservoir.

In another aspect of this embodiment, the system comprises a pluralityof fluid reservoirs each housing a fluid composition comprising theionic target analyte and an ionic liquid, wherein any two of the fluidcompositions may be the same or different, and/or any two of the targetanalytes may be the same or different. The plurality of reservoirs maybe arranged in an array and/or contained within a substrate thatcomprises an integrated multiple reservoir unit. In a related aspect ofthe embodiment, the system further includes a means for positioning theejector in acoustic coupling relationship with respect to each of thefluid reservoirs in succession.

In a related aspect of the embodiment, the target analyte comprises abiomolecule.

In another embodiment, a method is provided for synthesizing andacoustically extracting a reaction product from a reaction mixture. Themethod comprises:

(a) providing, in a fluid reservoir, a reaction mixture containing afirst reactant, a second reactant, and a fluid medium, where thereaction mixture typically has a volume in the range of about 1 nL toabout 3 mL;

(b) subjecting the reaction mixture to a reaction condition that causesa chemical reaction between the first reactant and the second reactantto give a reaction product, where the fluid medium comprises a firstliquid in which the reaction product has a first solubility;

(c) mixing into the reaction mixture a second liquid that issubstantially immiscible with the first liquid and in which the reactionproduct has a second solubility that differs from the first solubilityby at least 50%, thereby providing a fluid composition having an upperlayer and a lower layer containing different concentrations of thereaction product; and

(d) applying focused acoustic energy to the fluid reservoir in a mannereffective to eject a fluid droplet containing the reaction product intoa droplet receiver.

In a related aspect of the aforementioned embodiment, the reactionmixture further includes a reaction catalyst. In another related aspect,the method results in the partitioning of the reaction product and thecatalyst into different liquids, thereby substantially separating thereaction product and the catalyst.

In another related aspect of the aforementioned embodiment, the reactionmixture further includes a surfactant. In another related aspect, themethod results in the partitioning of the reaction product and thesurfactant into different liquids, thereby substantially separating thereaction product and the surfactant.

In a further embodiment, the invention provides a method for thesynthesis and acoustic transfer of a reaction product, the methodcomprising:

(a) providing, in a fluid reservoir, a reaction mixture comprised of afirst reactant, a second reactant, and a fluid medium, the reactionmixture typically having a volume in the range of about 1 nL to about 3mL;

(b) subjecting the reaction mixture to a reaction condition that causesa chemical reaction between the first reactant with the second reactantto give a reaction product; and

(c) applying focused acoustic energy to the fluid reservoir in a mannereffective to eject a fluid droplet containing the reaction product intoa droplet receiver.

The invention additionally provides, in another embodiment, a method fordetermining a distribution coefficient D of an analyte in a mixture oftwo solvents, wherein the method comprises:

(a) combining, in a fluid reservoir, a known quantity X of an analytewith a first volume V₁ of a first solvent and a second volume V₂ of asecond solvent that is substantially immiscible with the first solvent,such that the analyte has a concentration X/(V₁+V₂) in the first solventand the second solvent combined,

thereby forming a two-phase fluid composition having an upper layer ofthe first solvent and a lower layer of the second solvent, wherein theanalyte has a concentration C₁ in the first solvent and a concentrationC₂ in the second solvent;

(b) acoustically ejecting a droplet of the upper layer;

(c) determining C₁ in the ejected droplet;

(d) calculating C₂ from C₁ according to the relationship C₂=(C₁V₁)/V₂;and

(e) determining the distribution coefficient D by ascertaining the ratioof C₁ to C₂.

In another embodiment, the invention provides an acoustic method fordetermining a distribution coefficient D of an analyte in a mixture oftwo solvents, where the quantity of analyte may or may not be known, andthe method comprises:

(a) combining, in a fluid reservoir, an analyte, a first volume V₁ of afirst solvent, and a second volume V₂ of a second solvent that issubstantially immiscible with the first solvent, thereby forming apartitioned fluid composition having an upper layer of the first solventand a lower layer of the second solvent, wherein the analyte has aconcentration C₁ in the first solvent and a concentration C₂ in thesecond solvent;

(b) acoustically ejecting a droplet of the upper layer;

(c) determining C₁ in the ejected droplet in (b);

(d) removing the upper layer from the partitioned fluid composition;

(e) acoustically ejecting a droplet of the lower layer;

(f) determining C₂ in the ejected droplet in (e); and

(g) determining the distribution coefficient D by ascertaining the ratioof C₁ to C₂.

In a further embodiment of the invention, an acoustic system forextracting a target analyte from a sample is provided, wherein thesystem comprises:

(a) a fluid reservoir housing a fluid composition, wherein the fluidcomposition is a reaction mixture comprised of a first reactant, asecond reactant, and a fluid medium, the reaction mixture having avolume in the range of about 1 nL to about 3 mL; and

(b) an acoustic droplet ejector in acoustic coupling relationship withthe fluid reservoir for generating acoustic radiation in a mannereffective to eject a fluid droplet from the fluid composition into adroplet receiver, the ejector comprising an acoustic radiation generatorand a focusing means for focusing the acoustic radiation at a focalpoint within the reservoir.

In an additional embodiment, the invention provides method for removingmetal ions from an aqueous sample, wherein the method comprises:

adding to an aqueous sample that comprises a target analyte and a metalion, a metal extraction composition that comprises an ionic liquidcomposed of (a) a positively charged crown ether, a positively chargedcryptand, or a combination thereof, and (b) a negatively chargedcounterion;

warming the initial binary phase solution until the two phases becomemiscible, thereby mixing the metal ion with the metal extractioncomposition in a single phase solution;

and

cooling the single phase solution to generate a second binary phasesolution comprising an upper aqueous layer and a lower layer of themetal extraction composition and the metal ion.

In a related embodiment, the invention provides a metal extractioncomposition for use in the aforementioned (or other) process, comprisingan ionic liquid of a positively charged crown ether, a positivelycharged cryptand, or a combination thereof, and a negatively chargedcounterion.

In another embodiment, the invention provides a liquid-liquid separationmethod, comprising:

(a) providing, in a fluid reservoir, a fluid composition that contains atarget analyte and a non-analyte component and comprises an upper layerand a lower layer, wherein (i) the target analyte is at a first analyteconcentration in the upper layer and at a second analyte concentrationin the lower layer, and (ii) the non-analyte component is at a firstcomponent concentration in the upper layer and at a second componentconcentration in the lower layer; and

(b) applying focused acoustic energy to the fluid reservoir in a mannereffective to eject a fluid droplet from the fluid composition into adroplet receiver.

In a further embodiment, a liquid-liquid separation method is providedthat comprises:

(a) providing a sample comprising a target analyte and a non-analytecomponent in a first fluid;

(b) combining the sample with a second fluid to provide a fluidcomposition;

(c) subjecting the fluid composition to a mixing condition;

(d) allowing the fluid composition to settle into a separated fluidcomposition comprising an upper layer and a lower layer, wherein thetarget analyte has an upper analyte concentration in the upper layer anda lower analyte concentration in the lower layer, and the non-analytecomponent has an upper concentration in the upper layer and a lowercomponent concentration in the lower layer, wherein either (i) the loweranalyte concentration and the upper analyte concentration are different,(ii) the lower component concentration and the upper componentconcentration are different, or both (i) and (ii).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates a representative method of theinvention in which lipidic components are extracted from a biologicalsample into an upper organic layer, followed by acoustic ejection of thelipidic layer.

FIG. 2 and FIG. 3 schematically illustrate another representative methodof the invention in which DNA is extracted from an aqueous buffer intoan ionic liquid, with the buffer thereafter acoustically removed (FIG.2) and the DNA in the ionic liquid then processed for further analysisusing an extraction buffer (FIG. 3).

FIG. 4 schematically illustrates a method of the invention in which DNAis removed from a biological sample using an ionic liquid in a two-stepextraction process.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions and Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which the invention pertains. Specific terminology of particularimportance to the description of the present invention is defined below.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, “a fluid” refers not only to asingle fluid but also to a combination of two or more different fluidsthat may or may not be combined, “a solvent” or “a liquid,” e.g., “anionic liquid,” refers to a single solvent or liquid as well as to two ormore solvents and two or more liquids, wherein the two or more solvents,or the two or more liquids, may be separate or combined.

The term “radiation” is used in its ordinary sense and refers toemission and propagation of energy in the form of a waveform disturbancetraveling through a medium such that energy is transferred from oneparticle of the medium to another without causing any permanentdisplacement of the medium itself. The radiation used in conjunctionwith the present acoustically based extraction methods and systems isacoustic radiation.

The terms “acoustic radiation” and “acoustic energy” are usedinterchangeably herein and refer to the emission and propagation ofenergy in the form of sound waves. As with other waveforms, acousticradiation may be focused using a focusing means, as discussed below.

The terms “focusing means” and “acoustic focusing means” refer to ameans for causing acoustic waves to converge at a focal point, either bya device separate from the acoustic energy source that acts like a lens,or by the spatial arrangement of acoustic energy sources to effectconvergence of acoustic energy at a focal point by constructive anddestructive interference. A focusing means may be as simple as a solidmember having a curved surface, or it may include complex structuressuch as those found in Fresnel lenses, which employ diffraction in orderto direct acoustic radiation. Suitable focusing means also includephased array methods as are known in the art and described, for example,in U.S. Pat. No. 5,798,779 to Nakayasu et al. and Amemiya et al. (1997)Proceedings of the 1997 IS&T NIP13 International Conference on DigitalPrinting Technologies, pp. 698-702.

The terms “acoustic coupling” and “acoustically coupled” used hereinrefer to a state wherein an object is placed in direct or indirectcontact with another object so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two items are indirectly acoustically coupled, an “acousticcoupling medium” is needed to provide an intermediary through whichacoustic radiation may be transmitted. Thus, an ejector may beacoustically coupled to a fluid, e.g., by immersing the ejector in thefluid or by interposing an acoustic coupling medium between the ejectorand the fluid to transfer acoustic radiation generated by the ejectorthrough the acoustic coupling medium and into the fluid.

The terms “fluid reservoir” and “reservoir,” as used herein, refer to areceptacle, chamber, or surface region for holding or containing afluid. Thus, a fluid in a reservoir necessarily has a free surface,i.e., a surface that allows a droplet to be ejected therefrom. In itsone of its simplest forms, a reservoir may be a location on a solidsurface that has sufficient wetting properties to hold a fluid within alocalized region solely as a result of contact between the fluid and thesurface, wherein the localized region serves as a reservoir.

The term “fluid,” as used herein, refers to matter that is at leastpartially liquid. A fluid may contain a solid that is minimally,partially or fully solvated, dispersed or suspended. Examples of fluidsinclude, without limitation, aqueous liquids (including water per se andsalt water); aqueous solutions; nonaqueous liquids such as organicsolvents and the like; nonaqueous solutions; colloids; suspensions;emulsions; and gels. The fluid may be a biological sample fluid in whichthe analyte of interest is just one component of many.

The term “moiety” as used herein refers to any particular composition ofmatter, e.g., a molecular fragment, an intact molecule (including amonomeric molecule, an oligomeric molecule, or a polymer), or a mixtureof intact molecules or other materials (for example, a mixture of DNA ofdifferent lengths and/or sequences).

The term “near” as used herein refers to the distance from the focalpoint of the focused acoustic radiation to the surface of the fluid fromwhich a droplet is to be ejected and indicates that the distance shouldbe such that the focused acoustic radiation directed into the fluidresults in droplet ejection from the fluid surface so that one ofordinary skill in the art will be able to select an appropriate distancefor any given fluid using straightforward and routine experimentation.Generally, however, a suitable distance between the focal point of theacoustic radiation and the fluid surface is in the range of about 1 toabout 15 times the wavelength of the acoustic radiation in the fluid(i.e., the acoustic radiation used to eject the droplet), more typicallyin the range of about 1 to about 10 times that wavelength, preferably inthe range of about 1 to about 5 times that wavelength.

The term “substantially” as in, for example, the phrase “substantiallyidentical reservoirs,” refers to reservoirs that do not materiallydeviate in acoustic properties. For example, acoustic attenuations of“substantially identical reservoirs” deviate by not more than 10%,preferably not more than 5%, more preferably not more than 1%, and mostpreferably at most 0.1% from each other. Other uses of the term“substantially” involve an analogous definition.

The “target analyte” (sometimes referred to herein as simply “analyte)in the fluid sample may be any moiety that is an analyte of interest.The analyte can be an atom, an ion, a salt, a molecule, a class ofmolecules with a common characteristic (e.g., lipids, or salts), wherethe molecule and molecular class include organic compounds, inorganiccompounds, and organometallic compounds. The analyte may be one that isrelevant in environmental work (e.g., pertaining to water qualityevaluation), in the pharmaceutical context, in the chemical industry, inthe energy field, and in numerous other areas. Representative examplesof analytes include, without limitation, drugs, metabolites, inhibitors,ligands, receptors, catalysts, synthetic polymers, metals, metal ions,dyes, pesticides (e.g., DDT, eldrin, tetrachlorodibenzodioxin [TCDD],etc.), carcinogens (e.g., polycyclic aromatic hydrocarbons [PCAHs]),allosteric effectors, antigens, and viruses (e.g., HIV, HPV, hepatitisA, B, C, D, E, F, or G, cytomegalovirus, Epstein-Barr virus, yellowfever, etc.). Target analytes can also be reaction products orintermediates in a multi-step reaction. In addition, target analytes canbe a moiety of interest in which an extraction process of the inventioninvolves the transfer of some fraction of the analyte from a first fluidinto a second fluid. Target analytes can also be a component to beremoved from a fluid, e.g., a contaminant.

Often, the analyte is a “biomolecule,” also referred to herein as a“biological molecule,” where those terms refer to any molecular entitythat is commonly found in cells and tissues, and may be naturallyoccurring, recombinantly produced, biologically derived, chemicallysynthesized in whole or in part, or chemically or biologically modified.The term encompasses, for example, nucleic acids; amino acids; peptides,including oligopeptides, polypeptides, proteins, and conjugates thereofwith non-peptide moieties, such as nucleoproteins and glycoproteins;saccharides, including monosaccharides, disaccharides, andpolysaccharides; lipidic moieties; and covalent or non-covalentconjugates of any two or more of the foregoing, such as nucleoproteins,glycoproteins, lipoproteins, peptidoglycans, mucopolysaccharides, andthe like. Representative examples of biomolecules include enzymes,receptors, glycosaminoglycans, neurotransmitters, hormones, cytokines,cell response modifiers such as growth factors and chemotactic factors,antibodies, vaccines, haptens, toxins, interferons, ribozymes,anti-sense agents, plasmids, DNA, and RNA.

“Nucleic acids” may be nucleosides or nucleotides per se, but may alsocomprise nucleosides and nucleotides containing not only theconventional purine and pyrimidine bases, i.e., adenine (A), thymine(T), cytosine (C), guanine (G) and uracil (U), but also protected formsthereof, e.g., wherein the base is protected with a protecting groupsuch as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl,and purine and pyrimidine analogs. Suitable analogs will be known tothose skilled in the art and are described in the pertinent texts andliterature. Common analogs include, but are not limited to,1-methyladenine, 2-methyladenine, N⁶-methyladenine,N⁶-isopentyl-adenine, 2-methylthio-N⁶-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluoro-uracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methyl-aminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine. In addition, the terms “nucleoside” and “nucleotide”include those moieties that contain not only conventional ribose anddeoxyribose sugars, but other sugars as well. Modified nucleosides ornucleotides also include modifications on the sugar moiety, e.g.,wherein one or more of the hydroxyl groups are replaced with halogenatoms or aliphatic groups, or are functionalized as ethers, amines, orthe like.

Nucleic acids also include oligonucleotides, wherein the term“oligonucleotide,” for purposes of the present invention, is generic topolydeoxyribo-nucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,and to other polymers containing nonnucleotidic backbones. Thus, anoligonucleotide analyte herein may include oligonucleotidemodifications, for example, substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalkyl phosphoramidates and aminoalkyl phosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.). There is no intended distinction in length between theterms “polynucleotide” and “oligonucleotide,” and these terms are usedinterchangeably. These terms refer only to the primary structure of themolecule. As used herein the symbols for nucleotides and polynucleotidesare according to the IUPAC-IUB Commission of Biochemical Nomenclaturerecommendations (Biochemistry 9:4022, 1970).

“Peptide” analytes (or “peptidic” analytes) encompass any structurecomprised of one or more amino acids, and thus include peptides,dipeptides, oligopeptides, polypeptides, and proteins. The amino acidsforming all or a part of a peptide analyte may be any of the twentyconventional, naturally occurring amino acids, i.e., alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine(Y), as well as non-conventional amino acids such as isomers andmodifications of the conventional amino acids, e.g., D-amino acids,non-protein amino acids, post-translationally modified amino acids,enzymatically modified amino acids, β-amino acids, constructs orstructures designed to mimic amino acids (e.g., α,α-disubstituted aminoacids, N-alkyl amino acids, lactic acid, β-alanine, naphthylalanine,3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, andnor-leucine), and other non-conventional amino acids, as described, forexample, in U.S. Pat. No. 5,679,782 to Rosenberg et al. Peptide analytesmay also contain nonpeptidic backbone linkages, wherein the naturallyoccurring amide —CONH— linkage is replaced at one or more sites withinthe peptide backbone with a non-conventional linkage such asN-substituted amide, ester, thioamide, retropeptide (—NHCO—),retrothioamide (—NHCS—), sulfonamido (—SO₂NH—), and/or peptoid(N-substituted glycine) linkages. Accordingly, peptide analytes caninclude pseudopeptides and peptidomimetics. Peptide analytes can be (a)naturally occurring, (b) produced by chemical synthesis, (c) produced byrecombinant DNA technology, (d) produced by biochemical or enzymaticfragmentation of larger molecules, (e) produced by methods resultingfrom a combination of methods (a) through (d) listed above, or (f)produced by any other means for producing peptides.

“Saccharides,” or “saccharidic analytes,” include, without limitation,monosaccharides, disaccharides, oligosaccharides, polysaccharides,mucopolysaccharides or peptidoglycans (peptido-polysaccharides),pseudopeptidoglycans, and the like, wherein monosaccharides, includingmonosaccharide units in disaccharides, oligosaccharides,polysaccharides, and the like, include hexoses, pentoses, and tetroses,and may be in D- or L-form, and further wherein the glycosidic linkagesbetween monosaccharide units may be either α-glycosidic linkages orβ-glycosidic linkages. Illustrative examples of saccharidic analytesinclude the monosaccharides fructose, glucose, dextrose, galactose,mannose, ribose, deoxyribose, allose, fucose, rhamnose, erythrose,threose, and glyceraldehyde; the disaccharides sucrose, lactose,maltose, lactulose, trehalose, cellobiose; the polysaccharides amylose,amylopectin, glycogen, cellulose, chitin, callose, laminarin,chrysolaminarin, xylan, and galactomannan; and the mucopolysaccharides(also referred to as glycosaminoglycans) chondroitin sulfate, dermatansulfate, keratan sulfate, heparin, heparan sulfate, and hyaluronan.

“Lipids,” or “lipidic analytes,” refer to hydrophobic or amphiphilicmolecules, and include the broad categories of fatty acids,phospholipids, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids, and polyketides. Representative examples of lipidicmaterials include, but are not limited to, the following: phospholipidssuch as phosphorylated diacyl glycerides, particularly phospholipidsselected from the group consisting of diacyl phosphatidylcholines,diacyl phosphatidylethanolamines, diacyl phosphatidylserines, diacylphosphatidylinositols, diacyl phosphatidylglycerols, diacyl phosphatidicacids, and mixtures thereof, wherein each acyl group contains about 10to about 22 carbon atoms and is saturated or unsaturated; fatty acidssuch as isovaleric acid, valeric acid, caproic acid, enanthic acid,caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid,palmitic acid, stearic acid, arachidic acid, behenic acid, lignocericacid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid;lower fatty acid esters comprising esters of the foregoing fatty acids,wherein the carboxylic acid group —(CO)—OH of the fatty acid is replacedwith an ester moiety —(CO)—OR wherein R is a C₁-C₃ alkyl moietyoptionally substituted with one or two hydroxyl groups; fatty alcoholscorresponding to the aforementioned fatty acids, wherein the carboxylicacid group of the fatty acid is replaced by a —CH₂OH group; glycolipidssuch as cerebroside and gangliosides; oils, including animal oils suchas cod liver oil and menhaden oil, and vegetable oils such as babassuoil, castor oil, corn oil, cottonseed oil, linseed oil, mustard oil,olive oil, palm oil, palm kernel oil, peanut oil, poppyseed oil,rapeseed oil, safflower oil, sesame oil, soybean oil, sunflower seedoil, tung oil, or wheat germ oil; and waxes, including animal waxes suchas beeswax, lanolin, and shellac wax; mineral waxes such as montan wax;petroleum-derived waxes such as microcrystalline wax and paraffin wax;and plant waxes such as carnauba wax and candelilla wax.

“Extraction” and “extracting,” as those terms are used herein, refers toa process that involves the migration of a target analyte from a firstfluid into a second fluid and thus encompasses a separation process asexplained in the previous section. The terms typically refer to theenhancement of one component of a composition, a target analyte,relative to other components of the composition, e.g., contaminants, inone of two fluidic phases. Extraction may be complete, meaning that thetarget analyte is completely separated from other components of asample, so that following extraction, one fluid phase contains 100% ofthe target analyte. Extraction may also be partial, in which case somefraction less than 100% of the target analyte is isolated from othercomponents in a composition. Accordingly, extraction using the presentmethod may be for the purpose of increasing or decreasing theconcentration of a target analyte in one of the fluids; removing some orall of the analyte from one of the fluids; concentrating the amount ofanalyte in one of the fluids; isolating the analyte; purifying theanalyte; removing components, e.g., contaminants, that are initiallyassociated with a target analyte; or a combination of two or more of theforegoing. “Liquid-liquid extraction” as the term is used herein refersto an extraction process in which the first fluid and the second fluidare independently selected from fluids that comprise liquids; as such,liquid-liquid extraction encompasses gel-liquid extraction,suspension-liquid extraction, and the like. The extraction process hereis coupled with an acoustic ejection process, such that fluid dropletsare acoustically ejected from a fluid having an increased or decreasedconcentration of a target analyte (relative to the initial concentrationof target analyte in a sample or in an initial, pre-extraction fluidlayer or fluid composition) or increased or decreased concentration of anon-target component (relative to the initial concentration of anon-target component in a sample or in an initial, pre-extraction fluidlayer or fluid composition).

Reference to a sample “containing” or “comprising” an analyte includesboth a sample known to contain an analyte, although the identity of theanalyte may be unknown, and a sample suspected of containing an analyte.

The term “array” as used herein refers to a two-dimensional arrangementof features, such as an arrangement of reservoirs (e.g., wells in a wellplate) or an arrangement of fluid droplets or molecular moieties on asubstrate surface (as in an oligonucleotide or peptide array).

Arrays are generally comprised of features regularly ordered in, forexample, a rectilinear grid, parallel stripes, spirals, and the like,but non-ordered arrays may be advantageously used as well. An arraydiffers from a pattern in that patterns do not necessarily containregular and ordered features. In addition, arrays and patterns formed bythe deposition of ejected droplets on a surface, as provided herein, areusually substantially invisible to the unaided human eye. Arraystypically, but do not necessarily, comprise at least about 4 to about10,000,000 features, generally in the range of about 4 to about1,000,000 features.

2. Extraction Methodology

The present invention makes use of acoustic droplet ejection (ADE) inthe extraction of a target analyte from a fluid composition. In oneembodiment, ADE is implemented in an extraction process to produce afluid droplet containing a target analyte at a selected concentration. Afluid composition containing the target analyte is provided in a fluidreservoir, where the fluid composition is composed of two or morephases. That is, the fluid composition includes an upper region, orupper layer, as well as one or more lower regions, or lower layers,e.g., the fluid composition may be composed of two layers, three layers,four layers, or five or more layers. For simplicity, the method will bedescribed with respect to a two-phase system, in which the fluidcomposition comprises an upper layer of a first fluid and a lower layerof a second fluid, where the first fluid and second fluid aresubstantially immiscible at the conditions employed for extraction. Thetarget analyte in the fluid composition is present in the first fluid ata first concentration, and in the second fluid at a second concentrationthat is different from the first concentration. Generally, the firstconcentration and the second concentration differ by at least about 50%,e.g., at least about 85%, such as at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, or more. Focused acoustic energy is applied to the fluidreservoir in a manner effective to eject a fluid droplet from the fluidcomposition, generally toward and into a droplet receiver. The selectedconcentration, i.e., the concentration of the target analyte in theejected fluid droplet, is substantially equivalent to either the firstconcentration or the second concentration. In addition, the selectedconcentration is substantially uniform throughout the droplet. In oneexample, some fraction of a target analyte is moved from one fluid(e.g., a fluid containing a biological sample such as a cell lysate orthe like) to a second fluid. In another example, the concentration oftarget analyte in two fluids may or may not change, but the componentsassociated with the target analyte in one fluid may not be present in asecond fluid after the extraction process. For instance,post-extraction, one fluid may contain target analyte and a plurality ofcontaminants, while a second fluid contains the target analyte withoutthe contaminants present, or with the concentration of the contaminantssubstantially reduced. As another example, the extraction processinvolves changing the concentration of a target analyte or othercomponent in one or more fluid layers. In a variation on theaforementioned example, extraction involves two target analytes, wherethe process results in an increased concentration of one of the targetanalytes and a decreased concentration of the other target analyte inone or more fluid layers.

As the present methodology is effective with very small sample sizes,the total fluid composition in the reservoir generally occupies a volumeof no more than about 125 μL, e.g., no more than about 60 μL, no morethan about 45 μL, no more than about 30 μL, and the like.

The fluid composition may comprise a sample that contains the targetanalyte, such as a biological sample. The biological sample may comprisea sample dissolved or suspended in a fluid, or the biological sampleitself may be fluidic. Biological samples include, by way of example,tissue, tissue homogenate, cells, cell suspensions, cell extracts, wholeblood, plasma, serum, saliva, sputum, nasal discharge, cerebrospinalfluid, interstitial fluid, lymph fluid, semen, vaginal fluid, or feces.More typical biological samples are comprised of tissue, cells, orblood. A biological sample may or may not be processed in some mannerprior to extraction; preliminary processing methods are known in the artand include, for example, incorporation of an anticoagulant into a bloodsample; separation of blood into plasma and serum; alternatingcentrifugation and resuspension procedures for various sample types;incorporation of a preservative or a preservative-containing transportmedium into a sample; and the like. The invention is not limited in thisregard, however, and is readily implemented with biological samples thathave not undergone any preliminary processing as well as non-biologicalsamples.

Target analytes in biological samples include, without limitation,proteins, peptides, peptide fragments, lipidic compounds, and nucleicacids, particularly DNA. Other biological target analytes and othertypes of target analytes are set forth in Part (1) of this section,“Definitions and Terminology.”

Extraction of a target analyte using two or more fluids relies on thedifferential affinity of the analyte for one fluid relative to anotherfluid. The term “affinity” includes any factor or combination of factorsthat cause an analyte to partition into one fluid relative to anotherfluid. Examples of such factors include, without limitation:compatibility of analyte and fluid with respect to degree of polarity;ionic interaction between analyte and fluid; hydrogen bonding betweenanalyte and fluid; relative hydrophilicity or hydrophobicity of analyteand solvent; and, more generally, and not unrelated to theaforementioned factors, the degree to which an analyte is soluble in afluid.

In some embodiments, the extraction process of the invention involvesuse of two liquids in which the solubility of the target analyte isdifferent, with the differential in solubilities corresponding to thedifferential in the first concentration and the second concentration asexplained above. The differential in solubilities may be at least about50%, e.g., at least about 85%, such as at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%. In some embodiments, the differential in solubilities maybe less than about 50%. In other embodiments, as alluded to above, thesolubility differential results from the use of a first liquid that isis relatively hydrophobic and a second liquid that is relativelyhydrophilic, such that a hydrophilic moiety will preferentiallypartition into the hydrophilic liquid, while a hydrophobic moiety willpreferentially partition into the hydrophobic liquid. Such a system isuseful, for instance, with a fluid composition comprising a plurality ofcomponents having different hydrophobicities, e.g., a sample containinga target analyte having a hydrophobicity that is different from thehydrophobicity of other components also present in the sample.

In other embodiments, the target analyte may be a polar molecule orsalt, in which case a relatively polar solvent, which may be eitherprotic or aprotic, is combined with a relatively non-polar solvent inthe extraction process. With targets that undergo hydrogen bonding,protic solvents are useful; in such a case, a protic solvent is combinedwith an aprotic solvent that may or may not be nonpolar.

In addition, some analytes, including, but not limited to, some ionicanalytes, can be readily extracted with ionic liquids, as will beexplained infra. In some instances, an ionic liquid is used to extractan ionic analyte from another fluid composition. In other instances, anionic liquid is used that causes an ionic analyte to preferentiallypartition into a second fluid composition that is not an ionic liquid.In some instances, both such processes are combined in a multistepextraction process as will be explained in further detail below.

A wide variety of solvents can be used in conjunction with theinvention. In fact, virtually any solvent can be used provided thatthere is no adverse effect on the extraction process of the invention.Of course, nontoxic solvents are preferred. The liquids used in a singleextraction step or process may vary in viscosity, volatility, and otherchemical and physical characteristics. Extraction of a target analytefrom a viscous material is facilitated using a relatively nonviscousfluid. When one of the solvents is volatile, concerns about evaporationcan be set aside by use of a second solvent that has a lower density andis less volatile; the less volatile, lower-density solvent will form anupper layer over the lower, more volatile solvent, preventingevaporation thereof.

Solvents that can be employed in the methods and systems of theinvention include aqueous and organic solvents, protic and aproticsolvents, ionic and non-ionic liquids, polymeric and nonpolymericliquids, and the like. In combination, the fluids can form a monophasic,biphasic, triphasic, or higher multiphasic fluid composition; with acombination of two fluids, the resulting fluid composition is monophasicor biphasic. In some embodiments, the invention makes use of the degreeto which the selected fluids are miscible as well as the conditionsunder which a combination of selected liquids can be rendered more orless miscible.

Examples of solvents useful in conjunction with the present inventioninclude, without limitation, acetic acid, acetone, acetonitrile,ammonia, benzene, n-butanol, i-butanol, 2-butanol, t-butanol,2-butanone, butyl acetate, t-butyl alcohol, carbon tetrachloride,chlorobenzene, chloroform, cyclohexane, cyclohexanone, cyclopentane,1,2-dichloroethane, dichloromethane, diethyl ether, diethylene glycol,diethylene glycol dimethyl ether (diglyme), di-isopropyl ether,1,2-dimethoxyethane (glyme, DME), dimethyl ether, dimethylformamide(DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate,ethylene glycol, ethyl formate, formic acid, furan, glycerin, heptane,hexamethylphosphoramide (HMPA), hexamethylphosphorous triamide (HMPT),n-hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride,methyl ethyl ketone (MEK), methyl formate, N-methyl-pyrrolidinone (NMP),nitromethane, 1-octanol, n-pentane, petroleum ether (ligroine),piperidine, polyethylene glycol, n-propanol, i-propanol, pyridine,tetrahydrofuran (THF), toluene, trichloroethylene, triethylamine (TEA),water, deuterium oxide, o-xylene, m-xylene, p-xylene, and other solventsdescribed in U.S. Pat. Nos. 6,548,308 and 6,642,061, both to Ellson etal., incorporated by reference herein. Preferred aqueous solventsinclude water, an aqueous solution of buffering compounds and/or salts,such as phosphate buffer, Tris buffer, MES buffer, HEPES buffer,ammonium bicarbonate, and the like.

Specific examples of useful solvent systems and solvent combinationsherein include, without limitation: an aqueous solvent such as water orbuffer solution and an organic solvent such as cyclohexane,dichloromethane, 1-octanol, n-pentane, n-butanol, or a perfluorinated orsemi-fluorinated alkane solvent such as perfluoroheptane,1,1,1,2,3,3-hexafluoropropane, pentafluoropentane, and the like; anaqueous solvent such as water or buffer solution and a lipidic solventsuch as an oil; a binary system with two aqueous layers, where theaqueous liquids contain different solutes and are immiscible—see, e.g.,Partitioning in Aqueous Two-Phase System: Theory, Methods, Uses, andApplications to Biotechnology, Eds. Harry Walter et al. (Academic Press,1985); Hamta et al. (2017), “Application of polyethylene glycol basedaqueous two-phase systems for extraction of heavy metals,” Journal ofMolecular Liquids 231:20-24; and Eiden et al. (2016), “Two-Phase SystemRehydration of Antibody-Polymer Microarrays Enables ConvenientCompartmentalized Multiplex Immunoassays,” Analytical Chemistry 88(23)(also see Tavana et al. (2010) Adv. Mater. 22(24):2628-2631 and Fang etal. (2012) Tissue Engineering Part C: Methods 18)9):647-657); acombination of two aqueous liquids that contain the same solute but havea different pH, as would be the case, for instance, with two aqueousliquids having the same buffer composition but where the first andsecond aqueous liquids are buffered to a different pH; and a binarysystem with two organic layers, where the organic liquids areimmiscible, e.g., an ethanol/cyclohexane combination, ahexane/dichloromethane combination, an ether/chloroform combination, orthe like;

The two fluids selected for the extraction process should be immiscibleunder the conditions employed for extraction. Miscibility, as isunderstood in the art, refers to the degree to which one fluid issoluble in another fluid. This solubility may vary with temperature,hydrostatic pressure, or other factors, and this variable can beadvantageously incorporated into the present process. That is, the fluidcomposition can be subjected to a condition that induces a phasetransition and renders the two fluids miscible, so that the targetanalyte is thoroughly mixed with both fluids. Thereafter, the fluidcomposition can be subjected to a condition that returns the two fluidsto an immiscible state, so that acoustic energy can target a focal zonein one of the two fluid layers and eject a fluid droplet formed from andprimarily comprising only one of the two layers. Miscibilities ofsolvents under various conditions can be determined by reference to thepertinent texts and literature, and/or can be determined empirically bycombining two solvents and ascertaining the degree of miscibility at arange of temperatures, pressures, or the like. Most commonly, themiscibility of two fluids herein is altered by a change in temperature(such that raising or lowering the temperature of a fluid compositioncontaining the two fluids alters their miscibility) and/or chemically,e.g., by addition of a salt such as sodium chloride or by a change inpH. In some cases, the two fluids employed in the present extractionprocesses are miscible between about 40° C. and about 90° C.,

It will be appreciated that the target analyte and the two fluids can bemixed prior to extraction using other techniques as well, such asrepeated inversion of a container housing the analyte and fluids,stirring, sonication, agitation, moderating the temperature of thefluids, ejection (e.g., acoustic ejection) of droplets of one fluidthrough the other fluid (as illustrated in FIG. 2, discussed infra), andthe like.

In one embodiment, the extraction method involves the use of an ionicliquid. As indicated in Part (1) of this section, an ionic liquidcomprises a salt in the liquid form. That is, an ionic liquid is largelyor entirely composed of ions (i.e., “substantially ionic,” meaning thatsubstantially all of the ionic liquid is ionic), in contrast to ordinaryliquids, which are predominantly made up of electronically neutralspecies. Preferred ionic liquids for use in conjunction with theinvention are purely ionic or substantially ionic, and are in a liquidstate at extraction conditions. More preferred ionic liquids are “roomtemperature ionic liquids” (RTILs). An RTIL is composed of a salt thathas a relatively low melting point and is in liquid form at temperaturesbelow 100° C., e.g., at a temperature in the range of about 0° C. toabout 100° C. RTILs are preferred herein, insofar as they offer theconvenience of facilitating an extraction process that can be carriedout without changing the temperature of the fluid composition. Ionicliquids are useful in the extraction of ionic analytes, e.g., negativelycharged analytes such as DNA, but are useful in the extraction of othertypes of analytes as well. Common ionic liquids include, withoutlimitation, imidazolium salts, pyrrolidinium salts, piperidinium salts,pyridinium salts, morpholinium salts, ammonium salts, phosphonium salts,sulfonium salts, and guanidinium salts. Suitable ionic liquids arelisted in, for example, the ionic fluid catalogs published bySigma-Aldrich (October 2012) and by EMD Chemicals Inc., and and can beacquired as commercially available products. Specific examples of ionicliquids are provided below. In the following list, anion abbreviationsare as follows:

Acetate (CH₃COO⁻), OAc;

Bis[oxalato(2-)]borate, bob;

Bis(trifluoromethylsulfonyl)imide, Tf₂N;

Dicyanamide, DCA;

Formate (HCO₂ ⁻), HCOO;

Hexafluorophosphate, PF₆ ⁻;

Hydrogensulfate, HSO₄ ⁻;

Hydroxyacetate (CH₂(OH)COO⁻), HOOAc;

Methanesulfonate (mesylate), CH₃SO₃ ⁻;

2-(2-Methoxyethoxy)ethyl sulfate, CH₃(OCH₂CH₂)₂OSO₄ ⁻;

Methylsulfate (CH₃—O—SO₃ ⁻), MeSO₄;

Octylsulfate (C₈H₇SO₄ ⁻), OcSO₄;

Nitrate, NO₃ ⁻;

Sulfamate, H₂NSO₃ ⁻;

Tetracyanoborate, B(CN)₄ ⁻;

Tetrafluoroborate, BF₄ ⁻;

Thiocyanate, SCN⁻;

p-Toluenesulfonate, or tosylate, Tos;

Tricyanomethane (C(CN)₃ ⁻), TCM;

Trifluoroacetate (CF₃COO⁻), TFA;

Trifluoromethanesulfonate (triflate, CF₃SO₃ ⁻), OTf; and

Tris(pentafluoroethyl) trifluorophosphate, FAP.

Representative ionic liquids, in which it is to be understood that thefirst indicated species is the cation and the second indicated speciesis the anion (with the positive and negative signs omitted to comportwith standardized ionic liquid nomenclature), include, withoutlimitation, the following:

1-Benzyl-methylimidazolium (Zmim) salts, such as [Zmim][Cl];

N,N-Bis(2-hydroxyethyl)butylammonium (HEBA) salts, such as [HEBA][Tf₂N]and [HEBA][HCOO];

Bis(2-hydroxyethyl)ammonium (HEA) salts, such as [HEA][TFA] and[HEA][OAc];

Bis(2-methoxyethyl)ammonium (MEA) salts, such as MEA sulfamate;

1-Butyl-2,3-dimethylimidazolium (Bmmim) salts, such as [Bmmim][Cl],[Bmmim][I], [Bmmim][PF₆], [Bmmim][BF₄], and [Bmmim][OTf];

1-Butyl-3-methylimidazolium (Bmim) salts, such as [Bmim] [Tf₂N], [Bmim][Cl], [Bmim][Br], [Bmim][I], [Bmim][DCA], [Bmim][PF₆], [Bmim][HSO₄],[Bmim][MeSO₄], [Bmim][OcSO₄], [Bmim][BF₄], [Bmim][B(CN)₄], [Bmim][Tos],[Bmim][TCM], [Bmim][TFA], [Bmim][NO₃], and [Bmim][OAc];

1-Butyl-3-methylpyridinium (B3mpy) salts, such as [B3mpy][Cl],[B3mpy][DCA], [B3mpy][MeSO₄], and [B3mpy][BF₄];

1-Butyl-4-methylpyridinium (B4mpy) salts, such as [B4mpy][Cl] and[B4mpy][BF₄];

1-Butyl-1-methylpyrrolidinium (Bmpyr) salts, such as [Bmpyr][bob],[Bmpyr][Tf₂N], [Bmpyr][Cl], [Bmpyr][DCA], [Bmpyr][OTf], [Bmpyr][FAP],and [Bmpyr][B(CN)₄];

N-Butylpyridinium chloride (Bpy) salts, such as [Bpy][Cl], [Bpy][PF₆],[Bpy][BF₄], and [Bpy][OTf];

N,N-Dimethyl(2-hydroxyethyl)ammonium (MMHEA) salts such as[MMHEA][HOOAc], [MMHEA][Tf₂N], and [MMHEA][TFA];

1,3-Dimethylimidazolium (Mmim) salts, such as [Mmim][Cl], [Mmim][Br],and [Mmim][MeSO_(4],)

1,1-Dimethylpyrrolidinium (MMpyr) salts such as [MMpyr][I] and[MMpyr][Tf₂N];

N-Dodecyl-N,N-dimethyl-3-sulfopropylammonium Tf₂N and OTf;

1-(2-Ethoxyethyl)-1-methylpyrrolidinium (EOEMpyr) salts such as[EOEMpyr][Tf₂N], [EOEMpyr][Br], [EOEMpyr][BCN₄,], [EOEMpyr][Tf₂N], and[EOEMpyr][FAP];

1-Ethyl-2,3-dimethylimidazolium (Emmim) salts such as [Emmim][Br],[Emmim][Cl], [Emmim][MeSO₄], and [Emmim][BF₄];

N-Ethyl-N,N-dimethyl-2-methoxyethylammonium Tf₂N, Br, B(CN)₄, and FAP;

N-Ethyl-N,N-dimethyl-propylammonium Tf₂N, Br, DCA,bis(trifluoromethylsulfonyl) imide (Nemmp tfn), bromide (Nemmp Br),dicyanamide (Nemmp DCN), B(CN)₄, and FAP;

1-Ethyl-3-methylimidazolium (Emim) salts such as [Emim][bob],[Emim][Tf₂N], [Emim][Br], [Emim][Cl], [Emim][DCA], [Emim][HSO₄],[Emim][MeSO₄], [Emim][OcSO₄], [Emim][B(CN)₄], [Emim][BF₄], [Emim][TFA],and [Emim][OTf];

Guanidium (gua) salts such as [gua][OTf] and [gua][FAP];

1-Hexadecyl-2,3-dimethylimidazolium (Cmmim) salts such as [Cmmim][Cl];

1-Hexadecyl-3-methylimidazolium (Cmim) salts such as [Cmim][Cl] and[Cmim][FAP];

1-Hexyl-1-methylpyrrolidinium (Hmpyr) salts such as [Hmpyr] [Tf₂N],[Hmpyr][FAP], and [Hmpyr][Cl];

1-Hexyl-2,3-dimethylimidazolium (Hmmim) salts such as [Hmmim][Cl] and[Hmmim][FAP];

1-Hexyl-3-methylimidazolium (Hmim) salts such as [Hmim][Tf₂N],[Hmim][Cl], [Hmim][PF₆], [Hmim][BF₄] [Hmim][OTf], and [Hmim][FAP];

N-Hexylpyridinium (HPy) salts such as [HPy][Cl], [HPy][Tf₂N],[HPy][OTf], and [HPy][FAP];

1-(2-Hydroxyethyl)-3-methylimidazolium (HOE-Mim) salts such as[HOE-Mim][Tf₂N], [HOE-Mim][Cl], [HOE-Mim][Br], [HOE-Mim][OTf], and[HOE-Mim][FAP];

N-(3-Hydroxypropyl)pyridinium (HOP-Py) salts such as [HOP-Py][Tf₂N],[HOP-Py][Cl], [HOP-Py][Br], [HOP-Py][B(CN₄)], and [HOP-Py][FAP];

1-(3-Methoxypropyl)-1-methylpiperidinium (MOPMpi) salts such as[MOPMpi][Tf₂N], [MOPMpi][Cl], [MOPMpi][Br], [MOPMpi][B(CN₄)], and[MOPMpi][FAP];

1-Methylimidazolium (Mim) salts such as [Mim][BF₄];

Methyltrioctylammonium [MOc₃A] salts such as [MOc₃A][Tf₂N],[MOc₃A][TFA], and [MOc₃A][OTf];

1-Octyl-3-methylimidazolium (Omim) salts such as [Omim][Cl], [Omim][I],[Omim][BF₄], and [Omim][FAP];

1-Octyl-1-methylpyrrolidinium (OMpyr) salts such as [OMpyr][Cl];

1-Propyl-3-methylimidazolium (Pmim) salts such as [Pmim][I];

1-(3-Sulfopropyl)-3-butylimidazolium Tf₂N and OTf;

N-(3-sulfopropyl)-pyridinium Tf₂N and FAP;

Tetrabutylammonium (NB4) salts such as [NB4][Tf₂N];

Tetramethylammonium (Nm4) salts such as [Nm4][bob] and [Nm4][FAP];

Trihexyl(tetradecyl)phosphonium (P(h3)t) salts such as [P(h3)t][bob],[P(h3)t][Tf₂N], [P(h3)t][PF₆], [P(h3)t][BF₄], [P(h3)t][DCA], and[P(h3)t][FAP];

1,2,3-Trimethylimidazolium (Mmmi) salts such as [Mmmi][I];

2-Amino-1,6-dimethylimidazo[4,5-b]-pyridine salts such as2-amino-1,6-dimethylimidazo[4,5-b]-pyridineTf₂N; and

Triethyl-hexadecylphosphonium (THP) salts such as [THP][DCN]. Ofparticular interest herein are [Bmim][Tf₂N], [Bmim][OAc], [B3mpy][Tf₂N],[B4mpy][Tf₂N], [MOc₃A][Tf₂N], [MMpyr][Tf₂N], and [P(h3)t][DCA].

Polymeric ionic liquids may also be used in conjunction with the presentinvention. Polymeric ionic liquids are known in the art and described,for instance, in Shaplov et al. (2011), “Polymeric Ionic Liquids:Comparisons of Polycations and Polyanions,” Macromolecules44(24):9792-9803; Wu et al. (2017), “Polymerizable ionic liquids andpolymeric ionic liquids: facile synthesis of ionic liquids containingethylene oxide repeating unit via methanesulfonate and theirelectrochemical properties,” RSC Advances 7: 5394-5401; and Mecerreyeset al. (2011), “Polymeric ionic liquids: Broadening the properties andapplications of polyelectrolytes,” Progress in Polymer Science 36(12):1629-1648. Also suitable for use herein are the thermo-responsivepoly(ionic liquid)-based nanogels described by Zhang et al. (2015)Molecules 20:17378-17392, the preparation of which is also described inthat publication.

It will be appreciated that the aforementioned list of ionic liquids ismerely illustrative and not intended to be limiting. Other ionic liquidsuseful herein include those described by Plechkova et al. (2008) Chem.Soc. Rev. 37:123-150; Branco et al., “Physico-Chemical Properties ofTask-Specific Ionic Liquids,” in Ionic Liquids: Theory, Properties, NewApproaches, Ed. A. Korkorin (Intech, 2011); and Plechkova et al. (2015),in Ionic Liquids Completely Uncoiled (Wiley, 2015). Still other ionicliquids useful in conjunction with the present methods are describedelsewhere in the literature and/or will be apparent to those of ordinaryskill in the art. Generally preferred ionic liquids for use hereinenable extraction of a significant fraction of a target analyte from asample (on the order of 50% or more, such as 50% to 100%, 50% to 95%,50% to 85%, 50% to 75%, etc.), and/or provide a fluid composition inwhich the concentration of at least one non-target component (e.g., atleast one contaminant) originally associated with the target analyte isdecreased in one of the fluid layers (e.g., decreased by at least 50%,such as 50% to 100%, 50% to 95%, 50% to 85%, 50% to 75%, etc.).Preferred ionic liquids are relatively non-toxic and easily used in thelaboratory and have a high affinity for the fluid reservoir surface soas to concentrate an aqueous extraction fluid in the reservoir center.In some cases, it may be desirable for a selected ionic liquid to havean auditable acoustic impedance. In addition, in some instances, anionic liquid that exhibits an extraction bias based on analyte size ispreferred, while in other instances a preferred ionic liquid is one thatdoes not exhibit an analyte size-related extraction bias (as is usuallythe case with nucleic acid analytes such as DNA).

In some embodiments, an ionic liquid used in the present extractionprocess is a magnetic ionic liquid. A magnetic ionic liquid serves as aliquid form of magnetic beads, and a magnetic ionic liquid containingthe target analyte of interest can be pulled to one side of the fluidreservoir to allow easy removal of the depleted non-ionic (e.g.,aqueous) layer. Magnetic ionic liquids (MILs) are known in the art andhave been described in the literature. See, e.g., Clark et al. (2015)Anal. Chem. 87:1552-1559. As described therein, examples of MILs includebenzyl trioctyl ammonium bromotrichloroferrate (III) and1,12-di-(3-hexadecyl-benzimidazolium) dodecanebis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate (III).

In one embodiment, the method is used in the extraction of a biomoleculefrom a biological sample, wherein the biomolecule preferentiallypartitions into a first liquid relative to a second liquid, and acousticejection technology is used to remove one of the two phases after mixingand subsequent partitioning is complete. For instance, and as will bedescribed in further detail infra, acoustic ejection technology can beimplemented so as to rapidly and successively eject droplets of ananalyte-containing upper fluid layer from a fluid reservoir, where theanalyte-containing fluid droplets are ejected into a droplet reservoirfor further processing and/or analysis. Acoustic ejection technology canalso be implemented so as to repeatedly eject droplets of an upper fluidlayer that does not contain analyte, thereby removing anon-analyte-containing upper fluid from the fluid reservoir and allowinganalyte to remain in the lower fluid layer.

An example of such a method is depicted in FIG. 1. In FIG. 1, abiological sample is provided in a fluid container. The sample is amulti-component mixture in an aqueous fluid. A second fluid is addedthat removes at least one component from the sample by virtue of thatcomponent's preference for the second fluid. The upper layer may then beremoved from the fluid container by decanting or, more preferably, byrepeated acoustic ejection using, e.g., a focused acoustic ejectionsystem as will be described infra. In the specific example illustratedin FIG. 1, it is desired to remove lipidic components from thebiological sample. Accordingly, the “first fluid” is the aqueous sampleitself, e.g., 50 μL of a cell assay (where the term “cell assay” is usedherein to refer to a cellular sample that may or may not have beenprocessed to some degree, such as a cell lysate), as shown in thefigure, containing nucleic acids, proteins, lipids, and othercomponents. As lipids are far more soluble in a nonpolar or low polaritysolvent, the selected “second fluid” is an organic solvent such as 10 μLof diethyl ether. Following this extraction process, thelipid-containing ether layer, on top of the aqueous layer, can beremoved as described above.

Interestingly, as may be seen in FIG. 1, combining two solvents withdifferent properties in a selected fluid reservoir can result ininversion of the meniscus. That is, prior to addition of a second fluid,the first fluid has a concave meniscus, but upon addition of a secondfluid the concave meniscus transforms into a convex shape at thefluid-fluid boundary. This may be desirable in multiple instances. Forexample, with the acoustic radiation focused at the central point in thefluid container, a lower fluid may be ejected through a central “liquidaperture” without contacting (or minimally contacting) the upper fluidlayer. This allows one to omit an upper layer removal step, streamliningthe overall extraction process. Meniscus reversal in this way may beaccomplished by using fluid reservoirs having an interior surface thatattracts or repels a particular fluid type, e.g., an organic solvent, anaqueous liquid, an ionic liquid, or the like. Fluid reservoirs with suchinterior surfaces can be obtained commercially, e.g., as microwellplates with different types of coatings, from numerous sources.

Another example of an extraction process of the invention will bedescribed with respect to the extraction of a biomolecule thatpreferentially partitions into an ionic liquid relative to a non-ionicliquid. An important such biomolecule is DNA, which may or may not bedouble-stranded DNA (dsDNA). The first steps of such a method are shownschematically in FIG. 2. A biological sample may be initially processedby lysing cellular and/or tissue material therein and then resuspendingthe sample in a suitable aqueous buffer (e.g., Tris buffer), referred toherein as the “initial buffer.” The biological sample may also comprisecirculating cell-free DNA. When carried out in a microwell plate, e.g.,a 384-well plate, a known quantity of an ionic liquid is added into eachof a plurality of wells, as the individual wells serve as fluidreservoirs in which extraction takes place. The ionic liquid is selectedso as to have a strong affinity for the DNA, and the initial buffer isselected so that the DNA preferentially partitions into the ionicliquid, i.e., relative to the buffer solution. As illustrated in FIG. 2,the method may involve adding 5 to 30 μl, e.g., 25 μl, of an ionicliquid such as [Bmim][PF₆] (1-butyl-3-methylimidazoliumhexafluorophosphate) into the individual wells. The individual wellillustrated in the figure is shown in an inverted configuration, becausethe well has preferably been filled with the ionic liquid byacoustically ejecting droplets upwards into the well using focusedacoustic ejection technology. Alternatively, a well containing a smallamount of an ionic liquid may simply be inverted, with surface tensionmaintaining the ionic liquid in place. The aqueous buffer containing theDNA is then introduced into each well, preferably, again, using focusedacoustic droplet ejection. The volume of the two liquids may or may notbe the same; in the figure, both volumes, i.e., the volume of the ionicliquid and the volume of the DNA-containing aqueous buffer, areindicated as 25 μl.

As indicated in FIG. 2, the density of the two liquids may differ. FIG.2 provides representative and non-limiting density numbers, with theionic liquid density indicated as being generally greater than 1.3 g/mLand the density of the aqueous DNA solution approximating 1.1 g/mL. As aresult, the two liquids in the inverted fluid reservoir will partitionwith the aqueous buffer representing the upper layer, on the “bottom” ofthe well, and the ionic liquid containing the DNA below the buffer, as alower layer. The well plate can then be inverted so that the relativepositioning of the two layers switches, as shown. That is, afterinversion, the ionic liquid containing the DNA is at the bottom of thewell, and the aqueous buffer layer has risen through and above the ionicliquid layer.

At this point, a further step is employed to remove the aqueous bufferlayer. This step makes use of a second aqueous buffer solution, referredto herein as an “extraction buffer.” In contrast to the initial buffer,the extraction buffer is selected so that the DNA preferentiallypartitions into the extraction buffer from the ionic liquid. The initialbuffer and the extraction buffer may contain different buffercomponents, or they may contain the same buffer components but havedifferent pH levels. The process is schematically illustrated in FIG. 3.In this case, the extraction buffer is introduced into the fluidreservoirs containing the ionic liquid with DNA therein, and the twoliquids mix. As microwells in a microwell plate can be readily used asfluid reservoirs, mixing can be accomplished simply by rapidly invertingthe well plate multiple times. Following mixing, and allowing time forseparation of the two phases, each reservoir contains the ionic liquidas a lower layer, and the extraction buffer, with DNA therein, as anupper layer. The upper layer—i.e., the aqueous DNA layer—can be removedusing any suitable means, although acoustic ejection of the layer ispreferred. Using acoustic ejection, the layer may be transferred intoinverted individual receptacles, as described above, or it may beacoustically ejected directly into an analytical instrument, e.g., amass spectrometer. Transfer of the aqueous DNA solution to theanalytical instrument using acoustic ejection can be carried out asdescribed in Sinclair et al. (2016) Journal of Laboratory Automation21(1):19-26 and U.S. Pat. No. 7,405,395 to Ellson et al. (Labcyte Inc.,San Jose, Calif.), both of which are incorporated by reference in theirentireties. Use of acoustic droplet ejection to transfer theDNA-containing aqueous liquid to an analytical instrument can beconducted with very little sample, on the order of several nanoliters,if necessary. In addition, acoustic transfer to the analyticalinstrument can be done very quickly, capable of generating over 10,000data points per hour, and is therefore ideally suited to high-throughputprocesses.

In another embodiment, an extraction method is provided using threedifferent fluids, wherein in a first step using a first fluid and asecond fluid the target analyte partitions into the second fluid, but ina second step using the second fluid and a third fluid, the targetanalyte partitions into the third fluid. That is, the target analytepreferentially partitions into the second fluid when the alternative isthe first fluid, but prefers the third fluid relative to the secondfluid. The first, second, and third fluids are selected so as to removemultiple species, e.g., contaminants or analytes for processingseparately, from the sample, thereby providing a purer solution of thetarget analyte in the third fluid. An example of this method is asfollows: An aqueous biological sample (i.e., the first fluid, containingDNA as the target analyte) is admixed with an ionic liquid (i.e., thesecond fluid, into which the target analyte will partition), and allowedto partition into an upper aqueous layer and a lower ionic liquid layer.The ionic liquid is selected so that the DNA will partition into thethat layer in this first step. The upper layer is then removed,preferably using acoustic ejection as described elsewhere herein,leaving a fluid composition comprising DNA, the target analyte, in theionic liquid. In a subsequent step, the ionic liquid containing the DNAis admixed with an aqueous extraction buffer having a pH selected sothat at least 60 wt. % of the DNA (and preferably at least 75 wt. %,such as at least 85 wt. %, at least 90 wt. %, at least 95 wt. %, or 100%of the DNA) will now move from the ionic liquid into the extractionbuffer, which forms an upper fluid layer on top of the ionic liquid. TheDNA-containing aqueous layer can then be acoustically ejected as before.

FIG. 4 schematically illustrates the aforementioned dual extractionmethod using 35 L of a biological sample (indicated as a cell lysate inthe figure) containing DNA as the target analyte. The biological sampleis contained in a reservoir that may be a stand-alone single reservoir,a stand-alone reservoir that is one of a group of other such reservoirs(e.g., a tube in a tube rack containing other tubes), a well in amicrowell plate, e.g., a 384-well plate, or the like. The “first fluid”may be an aqueous buffer containing DNA, as indicated in the figure. Anionic liquid is added to the fluid reservoir, forming a lower layerunderneath the aqueous phase. The two fluid layers are mixed using asuitable method; heating is preferred, as indicated in FIG. 4. Heatingnot only mixes the layers and renders them miscible (providing asuitable ionic liquid is selected that can be rendered miscible with anaqueous fluid at a relatively mild temperature), but also lyses thecells in the biological sample, releasing cell contents. The DNA willstill preferentially remain in the ionic liquid, provided that the firstfluid is buffered to a suitable pH, i.e., a pH at which the DNA willpartition into the ionic liquid instead of remaining in the aqueousphase. The upper fluid containing unwanted cellular components isremoved, by decanting, aqueous ejection, or any other suitable method;again, a preferred method involves focused acoustic ejection. Afterremoval of the aqueous phase, a single phase of ionic liquid containingthe target analyte remains in the fluid reservoir. An extraction bufferis then added and the two phases are mixed. In one embodiment, theextraction buffer, as explained above, is buffered to a pH selected sothat at least 60 wt. % of the DNA (and preferably at least 75 wt. %,etc., of the DNA, as above) may move from the ionic liquid into theextraction buffer, which forms an upper aqueous base. The DNA-containingaqueous fluid layer is then acoustically ejected as before.

3. Reaction Products

As explained earlier herein, the target analyte is not limited toanalytes contained in or derived from biological samples. The targetanalyte may be an inorganic compound, an organometallic compound, oreven an atom or ion; see part (1) of this section. In a furtherembodiment, then, the target analyte can be a reaction product in amulti-step reaction. That is the invention additionally provides amethod for the synthesis and acoustic extraction of a reaction product,where the method comprises: providing, in a fluid reservoir, a reactionmixture of a first reactant, a second reactant, and a fluid mediumcomprising a first liquid; subjecting the reaction mixture to acondition that causes a chemical reaction to occur between the firstreactant and the second reactant to give a reaction product having afirst solubility; mixing into the reaction mixture a second liquid thatis immiscible with the first liquid and in which the reaction producthas a second solubility that differs from the first solubility by atleast 50%, thereby providing a fluid composition having an upper layerand a lower layer containing different concentrations of the reactionproduct; and applying focused acoustic energy to the fluid reservoir ina manner effective to eject a fluid droplet containing the reactionproduct into a droplet reservoir. The reaction mixture may furtherinclude a reaction catalyst, a surfactant, or additional usefulcomponents. When a reaction catalyst and/or surfactant is used, the twoliquids can be selected so that the reaction product partitions into onelayer and the catalyst and/or surfactant partitions into the otherlayer, enabling removal of these other moieties from the reactionproduct. In a variation on the aforementioned process, the reactionproduct does not necessarily partition into one of the two layers, butthe other components, i.e., the catalyst and surfactant (which may beviewed as contaminants relative to the reaction product) do have anaffinity for one layer relative to the other layer. The extractionprocess in this case thus results in the movement of the contaminantsfrom one layer to another without concomitant movement of the targetanalyte, so that both layers contain target analyte but one of thoselayers has a much lower concentration of contaminants.

The reaction condition is generally, although not necessarily, selectedfrom the group consisting of: allowing the reaction to proceed in thereaction mixture for a predetermined reaction time; admixing thereactants; changing the temperature of the reaction mixture; adding atleast one catalyst to the reaction mixture; adding at least onesurfactant to the reaction mixture; introducing at least one additionalreactant into the reaction mixture; and two or more of the foregoing incombination.

In a related aspect of the invention, a method is provided for thesynthesis and acoustic transfer of a reaction product. The methodincludes the following steps:

(a) providing, in a fluid reservoir, a reaction mixture comprised of afirst reactant, a second reactant, and a fluid medium, the reactionmixture having a volume in the range of about 1 nL to about 3 mL;

(b) subjecting the reaction mixture to a reaction condition that causesa chemical reaction between the first reactant with the second reactantto give a reaction product; and

(c) applying focused acoustic energy to the fluid reservoir in a mannereffective to eject a fluid droplet containing the reaction product intoa droplet receiver.

The reaction condition is as above, i.e., selected from the groupconsisting of: allowing the reaction to proceed in the reaction mixturefor a predetermined reaction time; admixing the reactants; changing thetemperature of the reaction mixture; adding at least one catalyst to thereaction mixture; adding at least one surfactant to the reactionmixture; introducing at least one additional reactant into the reactionmixture; and two or more of the foregoing in combination.

In a related embodiment, an acoustic system is provided for extracting atarget analyte from a sample, where the system comprises: (a) a fluidreservoir housing a fluid composition, wherein the fluid composition isa reaction mixture comprised of a first reactant, a second reactant, anda fluid medium, the reaction mixture having a volume in the range ofabout 1 nL to about 3 mL; and (b) an acoustic droplet ejector inacoustic coupling relationship with the fluid reservoir for generatingacoustic radiation in a manner effective to eject a fluid droplet fromthe fluid composition into a droplet receiver, the ejector comprising anacoustic radiation generator and a focusing means for focusing theacoustic radiation at a focal point within the reservoir. In oneembodiment, the droplet receiver is an inverted fluid reservoir, such asa well in an inverted microwell plate. In another embodiment, thedroplet receiver is an analytical instrument, such as a massspectrometer, and the droplets ejected into the mass spectrometer—eitherdirectly or indirectly—are analyzed by the instrument.

4. Extraction of Metal Ions

In another embodiment, an extraction method is provided for removingmetal ions such as alkali metal ions and alkaline earth metal ions froman aqueous sample that contains a target analyte and at least one alkalimetal ion, typically lithium cations, sodium cations, or potassiumcations, and/or at least one alkaline earth metal ion, such as calciumor magnesium ions. This is particularly useful in the mass spectrometrycontext, in which an aqueous fluid containing a target analyte ofinterest is transferred (e.g., via acoustic ejection) into a massspectrometer for analysis. Ion suppression, i.e., suppression of targetanalyte ionization, is a well-known problem in mass spectrometry, andone of the most common causes of ion suppression is the presence ofsignificant amounts (on the order of 10⁻⁵ M or higher) of alkali metalcations and/or alkaline earth metal cations. See Volmer et al. (2006)LCGC North America 24(5): 498-510. Because of their biologicalrelevance, sodium and potassium are the two alkali metals of greatestconcern. And while polyvalent cations can generally be removed fromsolution by precipitation to form insoluble salts (as with theprecipitation of iron or copper by phosphate), monovalent alkali metalcations are more difficult to remove from solution. To date, crownethers, although tightly binding alkali metal ions, have not beeneffectively used to remove alkali metals from aqueous solutions becausean effective extraction process for separating the crown ether from theremainder of a sample had not yet been developed.

Accordingly, in this embodiment, a new method is provided thateffectively extracts alkali metals and/or alkaline earth metals frombiological samples and other aqueous compositions. The method involvesadding to an aqueous sample that comprises a target analyte and analkali metal ion and/or alkaline earth metal ion, a metal extractioncomposition that comprises an ionic liquid and a metal binding compoundselected from crown ethers, cryptands, and combinations thereof, therebyforming an initial binary phase solution; applying a condition to thebinary phase solution so that the two phases become miscible (e.g.heating), thereby mixing the metal salt with the metal extractioncomposition in a single phase solution; and regenerating a binary phasesolution, e.g., by cooling, where the binary phase solution generated inthis step comprises an upper aqueous layer and a lower layer of themetal extraction composition and the metal ion. The aqueous layer maythen be ejected into a mass spectrometer (or other type of dropletreceiver) for analysis of the target analyte. The metal extractioncomposition comprises the ionic liquid and the metal binding compound ina weight ratio in the range of about 1:100 to about 100:1, moretypically in the range of about 1:10 to about 10:1.

Preferred metal binding compounds are crown ethers such as12-crown-4,15-crown-5, and 18-crown-6. A particularly preferred metalbinding compound comprises a crown ether that has been converted to anionic liquid by functionalization with a cationic moiety, typically,although not necessarily, with a cationic moiety that corresponds to acation contained within the liquid salts enumerated in Part 2 of thissection, “Extraction Methodology.” By way of example rather thanlimitation, modification of a crown ether in this way can beaccomplished by replacing a hydrogen atom (within a C—H group) with afunctional group containing a positively charged nitrogen atomassociated with a negatively charged counterion. One such crown ethersalt has the structure of formula (I)

where R may be selected from tertiary amino groups and nitrogenheterocycles, and X is an anionic species such as a halide ion. Aspecific example of such a crown ether salt has the structure of formula(II)

This crown ether salt may be synthesized from chloromethyl 18-crown-6and N-methylimidazole using a technique analogous to that described byDharaskar et al. (2016), “Synthesis, characterization and application of1-butyl-3-methylimidazolium tetrafluoroborate for extractivedesulfurization of liquid fuel,” Arabian Journal of Chemistry 9:578-587.

In a related embodiment, the invention provides a metal extractioncomposition for use in the aforementioned extraction (or other) process,wherein the composition comprises an an ionic liquid and a metal bindingcompound selected from crown ethers, cryptands, and combinationsthereof. It should be noted that these functionalized crown ethers andcryptands can serve a dual purpose in the present extraction processes,in that they are ionic liquids with selective affinity for certain typesof target analytes, as well as chelating agents for removal of alkalimetal ions and alkaline earth metal ions.

5. Determination of a Distribution Coefficient of an Analyte in TwoSolvents

In any extraction process, including those described and claimed herein,it is extremely useful to know the distribution coefficient of aparticular target analyte in two solvents. One of the solvents may bewater, as in an aqueous biological sample, an aqueous buffer, or thelike, and the other solvent may be a candidate solvent underconsideration for use in an extraction process. Accordingly, in afurther embodiment, the invention provides a method for determining adistribution coefficient D of an analyte in a mixture of two solvents,where the method comprises the following steps:

(a) combining, in a fluid reservoir, a known quantity X of an analytewith a first volume V₁ of a first solvent and a second volume V₂ of asecond solvent that is substantially immiscible with the first solvent,such that the analyte has a concentration X/(V₁+V₂) in the first solventand the second solvent combined, thereby forming a partitioned fluidcomposition having an upper layer of the first solvent and a lower layerof the second solvent, wherein the analyte has a concentration C₁ in thefirst solvent and a concentration C₂ in the second solvent;

(b) acoustically ejecting a droplet of the upper layer;

(c) determining C₁ in the ejected droplet;

(d) calculating C₂ from C₁ according to the relationship C₂=(C₁V₁)/V₂;and

(e) determining the distribution coefficient D by ascertaining the ratioof C₁ to C₂.

In a related embodiment, a method is provided for determining thedistribution coefficient D in two solvents, comprising:

(a) combining, in a fluid reservoir, an analyte, a first volume V₁ of afirst solvent, and a second volume V₂ of a second solvent that issubstantially immiscible with the first solvent, thereby forming apartitioned fluid composition having an upper layer of the first solventand a lower layer of the second solvent, wherein the analyte has aconcentration C₁ in the first solvent and a concentration C₂ in thesecond solvent;

(b) acoustically ejecting a droplet of the upper layer;

(c) determining C₁ in the ejected droplet in (b);

(d) removing the upper layer from the partitioned fluid composition;

(e) acoustically ejecting a droplet of the lower layer;

(f) determining C₂ in the ejected droplet in (e); and

(g) determining the distribution coefficient D by ascertaining the ratioof C₁ to C₂.

6. Acoustic Ejection and High-Throughput Processes

Acoustic ejection enables rapid processing as well as generation ofnanoliter-sized droplets of predetermined and consistent size; see U.S.Pat. No. 6,416,164 to Stearns et al., incorporated by reference earlierherein. The aforementioned patent describes how the size of acousticallyejected droplets from a fluid surface can be carefully controlled byvarying the acoustic power, the acoustic frequency, the toneburstduration, and/or the F-number of the focusing lens, with lenses havingan F-number greater than approximately 2 generally preferred. ADE thusenables ejection of “ultra-monodisperse” droplets, which in the contextof the present invention means that the ejected particles have aconsistent diameter, with a coefficient of variation of about 1%. Thisin turn enables introduction of a fluid sample in a precise andpredetermined amount into the the system for analysis. An additionaladvantage of using acoustic ejection is that droplets can be ejectedfrom a very small sample size, on the order of 5 μl or less. This isparticularly advantageous when sample availability is limited and asmall fluid sample must be analyzed out of necessity. In terms ofprocessing capability, U.S. Pat. No. 6,938,995 to Mutz et al. explainsthat acoustic ejection technology, used in conjunction with acousticassessment of fluid samples in a plurality of reservoirs, can achieveanalysis of over 5, 10, or even 25 reservoirs per second, translating towell in excess of 50,000 fluid samples per day.

Because of the precision that is possible using acoustic ejectiontechnology, the present system can be used to acoustically eject samplefluid droplets of very small size. The invention is not limited in thisregard, however, and the volume of acoustically ejected droplets canrange from about 0.5 pL to about 3 mL. For many applications, the systemof the invention is used to generate nanoliter-sized fluid droplets foranalysis, where “nanoliter-sized” droplets generally contain at mostabout 30 nL of fluid sample, typically not more than about 10 nL,preferably not more than about 5.0 nL, more preferably not more thanabout 3.0 nL, such as not more than 1.0 nL, not more than about 50 pL,not more than about 25 pL, and not more than about 1 pL, includingranges of about 0.5 pL to 2.0 nL, about 0.5 pL to 1.5 nL, about 0.5 pLto 1.0 nL, about 1.0 pL to 2.0 nL, about 1.0 pL to 1.5 nL, about 1.0 pLto 1.0 nL, and the like. The typical operating range produces dropletsin the range of about 1 nL to about 30 nL. Acoustic ejection of dropletsfrom the surface of a fluid sample is carried out using an acousticejector as will be described in detail below. Acoustic ejectiontechnology is particularly suited to high-throughput processing,particularly high-throughput mass spectrometry (HTMS), insofar as HTMShas been hampered by the lack of easily automated sample preparation andloading, the need to conserve sample, the need to eliminate crosscontamination, the inability to go directly from a fluid reservoir intothe analytical device, and the inability to generate droplets of theappropriate size.

In one embodiment, then, the system and method of the invention make useof an acoustic ejector as a fluid sample droplet generation device toeject droplets from a fluid composition in the context of liquid-liquidextraction. The acoustic ejector directs acoustic energy into areservoir housing an analyte-containing fluid composition in a mannerthat causes ejection of a fluid droplet upward from the surface of thefluid.

The system may also include a means for positioning the reservoir andthe acoustic ejector in acoustic coupling relationship. Typically, asingle ejector is used that is composed of an acoustic radiationgenerator and a focusing means for focusing the acoustic radiationgenerated by the acoustic radiation generator. However, a plurality ofejectors may be advantageously used as well. Likewise, although a singlereservoir may be used, the device typically includes a plurality ofreservoirs, e.g., as an array. When the system is used to eject adroplet of an analyte-containing fluid sample from each of a pluralityof reservoirs, a positioning means is incorporated in order to move asubstrate containing the reservoirs (which may be positioned on amovable stage, for instance) relative to the acoustic ejector, or viceversa. Rapid and successive acoustic ejection of a fluid droplet fromeach of a series of reservoirs is thereby readily facilitated. Eithertype of positioning means, i.e., an ejector positioning means or areservoir or reservoir substrate positioning means, can be constructedfrom, for example, motors, levers, pulleys, gears, a combinationthereof, or other electromechanical or mechanical means.

While any acoustic droplet ejection system can be used in conjunctionwith present system and method, preferred ADE systems are thosedescribed in the following U.S. patents, all of common assignmentherewith and incorporated by reference herein: U.S. Pat. No. 6,416,164to Steams et al.; U.S. Pat. No. 6,666,541 to Ellson et al.; U.S. Pat.No. 6,603,118 to Ellson et al.; U.S. Pat. No. 6,746,104 to Ellson etal.; U.S. Pat. No. 6,802,593 to Ellson et al.; U.S. Pat. No. 6,938,987to Ellson et al.; U.S. Pat. No. 7,270,986 to Mutz et al.; U.S. Pat. No.7,405,395 to Ellson et al.; and U.S. Pat. No. 7,439,048 to Mutz et al.Preferred ADE systems for use herein are those available from LabcyteInc., particularly the Echo® 500-series Liquid Handler systems,including the Echo® 525, the Echo® 550, and the Echo® 555 LiquidHandlers, as well as the Echo® 600-series Liquid Handler systems,including the Echo® 600 and the Echo® 655 Liquid Handlers, all of whichcan eject a broad range of fluid classes with high accuracy, precisionand speed.

As described in the above patents, an acoustic ejection device may beconstructed to eject fluid droplets from a single reservoir or frommultiple reservoirs. To provide modularity and interchangeability ofcomponents, it may sometimes be preferred for the device to be used inconjunction with a plurality of removable reservoirs, e.g., tubes in arack or the like. Generally, the reservoirs are arranged in a pattern oran array to provide each reservoir with individual systematicaddressability. In addition, while each of the reservoirs may beprovided as a discrete or stand-alone container, in circumstances thatrequire a large number of reservoirs, it is preferred that thereservoirs are contained within an integrated multiple reservoir unit.As an example, the multiple reservoir unit may be a solid surface onwhich discrete fluid-containing regions are maintained in place byvirtue of surface wetting properties, with each localizedfluid-containing region constituting a reservoir. As another example,the multiple reservoir unit may be a well plate with the individualwells serving as reservoirs. Many well plates suitable for use with thedevice are commercially available and may contain, for example, 96, 384,1536, or 3456 wells per well plate, and having a full skirt, half skirt,or no skirt. Well plates or microtiter plates have become commonly usedlaboratory items. The Society for Laboratory Automation and Screening(SLAS) has established and maintains standards for microtiter plates inconjunction with the American National Standards Institute. The wells ofsuch well plates are generally in the form of rectilinear arrays.

The availability of such commercially available well plates does notpreclude the manufacture and use of custom-made well plates in othergeometrical configurations containing at least about 10,000 wells, or asmany as 100,000 to 500,000 wells, or more. Furthermore, the materialused in the construction of reservoirs must be compatible with the fluidsamples contained therein. Thus, if it is intended that the reservoirsor wells contain an organic solvent such as acetonitrile, polymers thatdissolve or swell in acetonitrile would be unsuitable for use in formingthe reservoirs or well plates. Similarly, reservoirs or wells intendedto contain DMSO must be compatible with DMSO. For water-based fluids, anumber of materials are suitable for the construction of reservoirs andinclude, but are not limited to, ceramics such as silicon oxide andaluminum oxide, metals such as stainless steel and platinum, andpolymers such as polyester, polypropylene, cyclic olefin copolymers(e.g., those available commercially as Zeonex© from Nippon Zeon andTopas® from Ticona), polystyrene, and polytetrafluoroethylene. Forfluids that are photosensitive, the reservoirs may be constructed froman optically opaque material that has sufficient acoustic transparencyfor substantially unimpaired functioning of the device.

In addition, to reduce the amount of movement and time needed to alignthe acoustic radiation generator with each reservoir or reservoir wellduring operation, it is preferable that the center of each reservoir belocated not more than about 1 centimeter, more preferably not more thanabout 1.5 millimeters, still more preferably not more than about 1millimeter and optimally not more than about 0.5 millimeter, from aneighboring reservoir center. These dimensions tend to limit the size ofthe reservoirs to a maximum volume. The reservoirs are constructed tocontain typically no more than about 1 mL, preferably no more than about100 pL, more preferably no more than about 1 pL, and optimally no morethan about 1 nL, of fluid. To facilitate handling of multiplereservoirs, it is also preferred that the reservoirs be substantiallyacoustically indistinguishable.

The acoustic ejection device used in conjunction with the present systemand method enables the acoustic ejection of droplets at a rate of atleast about 250 Hz, but higher ejection rates including 500 Hz, 1 kHz,or higher are possible, with smaller droplets enabling higher repetitionrates. The device is also capable of rapidly ejecting droplets from eachof a plurality of reservoirs, which may be arranged in array such as isthe case with a well plate or a rack of individual tubes. That is, asubstrate positioning means or an ejector positioning means acousticallycouples the ejector to each of a series of fluid reservoirs in rapidsuccession, thereby allowing fast and controlled ejection of fluidsample droplets from different reservoirs. Current commerciallyavailable technology allows for the substrate to be moved relative tothe ejector, and/or for the ejector to be moved from one reservoir toanother within the same substrate, with repeatable and controlledacoustic coupling at each reservoir, in less than about 0.1 second forhigh performance positioning means and in less than about 1 second forordinary positioning means. As explained in U.S. Pat. No. 6,666,541 toEllson et al., a custom designed system can reduce thereservoir-to-reservoir transition time (equivalent to the time betweenacoustic ejection events) to less than about 0.001 second. In order toprovide a custom designed system, it is important to keep in mind thatthere are two basic kinds of motion: pulse and continuous. Pulse motioninvolves the discrete steps of moving a substrate or an ejector intoposition so that the ejector is acoustically coupled to a reservoirwithin the substrate, acoustically ejecting a droplet from a samplefluid in the reservoir, and repositioning the substrate and/or ejectorso that the ejector is acoustically coupled to the next reservoir. Usinga high performance positioning means with such a method allowsrepeatable and controlled acoustic coupling at each reservoir in lessthan 0.1 second. A continuous motion design, on the other hand, movesthe substrate and/or ejector continuously, although not at the samespeed, and provides for ejection during movement. Since the pulse widthis very short, this type of process enables over 10 Hz reservoirtransitions, and even over 1000 Hz reservoir transitions.

The methodology of the invention is thus ideal for implementing thedisclosed extraction processes in the high-throughput context.Extraction can be carried out as described herein in each of asuccession of fluid reservoirs, e.g., wells in a microwell plate, withvery rapid reservoir-to-reservoir transitions and acoustic dropletejection into any type of droplet reservoir, e.g., an inverted microwellplate or an analytical instrument.

A representative focused acoustic ejection system that can beadvantageously used herein is illustrated in FIG. 1 of U.S. Pat. No.6,666,541 to Ellson et al., the disclosure of which is incorporated byreference. As explained therein, an acoustic droplet ejection devicecomprises an acoustic ejector, which includes an acoustic radiationgenerator and a focusing means for focusing the acoustic radiationgenerated at a focal point within a fluid sample, near the fluidsurface. The acoustic ejector is thus adapted to generate and focusacoustic radiation so as to eject a droplet of fluid from a fluidcomposition in a fluid reservoir. The acoustic radiation generator andthe focusing means may function as a single unit controlled by a singlecontroller, or they may be independently controlled. Any of a variety offocusing means that include curved surfaces or Fresnel lenses known inthe art may be employed in conjunction with the present invention. Suchfocusing means are described in U.S. Pat. No. 4,308,547 to Lovelady etal. and U.S. Pat. No. 5,041,849 to Quate et al., as well as in U.S.Patent Application Publication No. 2002037579. In addition, there are anumber of ways to acoustically couple the ejector to each individualreservoir and thus to the fluid therein. Although acoustic coupling canbe achieved through direct contact with the fluid contained in thereservoirs, the preferred approach is to acoustically couple the ejectorto the reservoirs and reservoir fluids without allowing any portion ofthe ejector (e.g., the focusing means) to contact any of the fluids tobe ejected.

The acoustic droplet ejector may be in either direct contact or indirectcontact with the external surface of each reservoir. With directcontact, in order to acoustically couple the ejector to a reservoir, itis preferred that the direct contact be wholly conformal to ensureefficient acoustic energy transfer. That is, the ejector and thereservoir should have corresponding surfaces adapted for mating contact.Thus, if acoustic coupling is achieved between the ejector and reservoirthrough the focusing means, it is desirable for the reservoir to have anoutside surface that corresponds to the surface profile of the focusingmeans. Without conformal contact, efficiency and accuracy of acousticenergy transfer may be compromised. In addition, since many focusingmeans have a curved surface, the direct contact approach may necessitatethe use of reservoirs that have a specially formed inverse surface.

Optimally, acoustic coupling is achieved between the ejector and each ofthe reservoirs through indirect contact, provided by an acousticcoupling medium placed between the ejector and the base of the fluidreservoir. The acoustic coupling medium may be an acoustic couplingfluid, preferably an acoustically homogeneous material in conformalcontact with both the acoustic focusing means and the underside of thereservoir. The system may contain a single acoustic ejector or it maycontain multiple ejectors. Single ejector designs are generallypreferred over multiple ejector designs because accuracy of dropletplacement and consistency in droplet size and velocity are more easilyachieved with a single ejector. However, the invention is not limited tosingle ejector designs.

When more than one fluid reservoir is used in the present methods, thereservoirs are preferably both substantially identical and substantiallyacoustically indistinguishable, although identical construction is not arequirement. As explained earlier in this section, the reservoirs may beseparate removable components in a tray, rack, or other such structure,but they may also be fixed within a plate, e.g., a microwell plate, orother substrate. Each reservoir is preferably substantially axiallysymmetric, as shown, having vertical walls extending upward fromcircular reservoir bases, although other reservoir shapes and reservoirbase shapes may be used. The material and thickness of each reservoirbase should be such that acoustic radiation may be transmittedtherethrough and into the fluid sample contained within each reservoir.

In operation, a fluid reservoir is filled with a fluid composition thatcomprises a sample containing a target analyte, as explained previously.The target analyte is generally in extracted form, in a solvent orsolvent mixture, or it may be in lower fluid layer as explained earlierherein. The acoustic ejector is positioned just below the fluidreservoir, with acoustic coupling between the ejector and the reservoirprovided. Once the ejector and reservoir are properly positioned withrespect to each other, the acoustic radiation generator is activated toproduce acoustic radiation that is directed by the focusing means to afocal point near a fluid surface within the reservoir (where a fluidsurface may represent either a liquid-air interface or a liquid-liquidinterface. As a result, a fluid droplet is ejected from the fluidsurface toward a droplet receiver such as a substrate, an invertedreservoir, a well in an inverted microwell plate, a droplet transportdevice, or an analytical instrument. In a multiple-reservoir system,e.g., a multiwell plate or tube rack, can then be repositioned relativeto the acoustic ejector such that another reservoir is brought intoalignment with the ejector and a droplet of the next fluid compositioncan be ejected.

An analytical instrument into which the ejected droplet may be directedcan be any instrument used for detecting a target analyte, determiningthe amount or concentration of target analyte in a sample, ordetermining the chemical composition of a target analyte. When theanalytical instrument is a mass spectrometer or other type of devicerequiring the analyte to be in ionized form, the exiting droplets passthrough an ionization region, prior to entering the mass spectrometer orother analytical instrument requiring that analyte be in ionized form.In the ionization region, a selected ionization source, e.g., anelectrospray ion source, converts the analyte to ionized form. Withejected fluid droplets that contain the target analyte in ionized form,exposure to an ionization source is unnecessary; see, e.g., ProvisionalU.S. Patent Application Ser. No. 62/590,079 for “System and Method forthe Acoustic Loading of an Analytical Instrument Using a Continuous FlowSampling Probe” to Datwani et al., filed Nov. 22, 2017, the disclosureof which is incorporated by reference herein. Exemplary analyticalinstruments include, but are not limited to, mass spectrometers,spectroscopy devices, separation systems, and combinations thereof.Exemplary ionization techniques include, but are not limited to,chemical ionization, electron impact ionization, desorption chemicalionization, inductively coupled plasma ionization, and atmosphericpressure ionization, including electrospray ionization and atmosphericpressure chemical ionization, and atmospheric pressure photo-ionization.Exemplary separation methods include, but are not limited to liquidchromatography, solid phase extraction, HPLC, capillary electrophoresis,or any other liquid phase sample cleanup or separation process.Exemplary mass spectrometers include, but are not limited to, sectormass spectrometers, time-of-flight mass spectrometers, quadrupole massfilter mass spectrometers, three-dimensional quadrupole ion trap massspectrometers, linear quadrupole ion trap mass spectrometers, toroidalion trap mass spectrometers, and Fourier transform ion cyclotronresonance mass spectrometers.

In addition, the invention herein encompasses modifications of theacoustic ejection process to optimize results, as previously described.For example, as explained in U.S. Pat. No. 6,932,097 to Ellson et al.,U.S. Pat. No. 6,938,995 to Ellson et al., U.S. Pat. No. 7,354,141 toEllson et al., U.S. Pat. No. 7,899,645 to Qureshi et al., U.S. Pat. No.7,900,505 to Ellson et al., U.S. Pat. No. 8,107,319 to Stearns et al.,U.S. Pat. No. 8,453,507 to Ellson et al., and U.S. Pat. No. 8,503,266 toStearns et al., the above acoustic droplet ejectors can be utilized forcharacterization of a fluid composition in a reservoir, e.g., to measurethe height of the fluid meniscus as well as other properties, such asfluid volume, viscosity, density, surface tension, composition, acousticimpedance, acoustic attenuation, speed of sound in the fluid, etc., anyor all of which can then be used to determine optimum parameters fordroplet ejection, including acoustic power, acoustic frequency,toneburst duration, and/or the F-number of the focusing lens. As anotherexample, acoustic interrogation processes can be used to optimize therelative position of the acoustic ejector and a fluid-containingreservoir in a focus-activated acoustic ejection system, as described inU.S. Pat. Nos. 8,544,976 and 8,882,226 to Ellson et al. An additionalexample is a method for optimizing the amplitude of the acousticradiation used to eject fluid droplets, by analyzing the waveforms ofacoustic radiation reflected from surfaces within the reservoir prior toejection; see U.S. Pat. Nos. 7,717,544 and 8,770,691 to Steams et al.Droplet size and consistency can be ensured using the method of U.S.Pat. No. 6,383,115 to Hadimioglu et al., and variations in reservoirproperties can be controlled for using the methods of U.S. Pat. No.7,481,511 to Mutz et al. and 7,784,331 to Ellson et al.

7. Dynamic Tracking of a Liquid-Liquid Boundary

In a preferred embodiment, a combination of the aforementioned methodsand systems for optimizing acoustic ejection processes can beadvantageously used in the present context. More specifically, thevertical position of the boundary between two fluid layers in a fluidreservoir can be tracked before each acoustic ejection event, as can theheight of the entire fluid composition in the reservoir. This can bedone using the acoustic interrogation techniques described in theaforementioned patents. During an extraction process of the invention,then, the height of the upper fluid (i.e., the distance from theliquid-liquid boundary to the meniscus, at the center point) can becalculated from the combination of the overall (central) height of thefluid composition and the vertical location of the (center of) theliquid boundary; that is, the height of the upper fluid equals theoverall fluid height minus the height of the identified boundary. This,in turn, facilitates a process in which acoustic ejection can be stoppedafter complete ejection of an upper layer without also ejecting a lowerlayer.

It is to be understood that while the invention has been described inconjunction with a number of specific embodiments, the foregoingdescription as well as the examples that follow are intended toillustrate and not limit the scope of the invention. In this regard, noattempt is made to show structural details of the invention in moredetail than is necessary for the fundamental understanding of theinvention, the description taken with the drawings and/or examplesmaking apparent to those skilled in the art how the invention may beembodied in practice. This disclosure includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theelements of the invention described herein are encompassed by thedisclosure unless otherwise indicated herein or clearly contradicted bycontext.

All patents, patent publications, literature references, and othermaterials cited herein are incorporated by reference in theirentireties.

Experimental

An Echo® 555 Liquid Handler (Labcyte Inc., San Jose, Calif.) serves asthe acoustic droplet ejector (ADE) system, as the system can eject abroad range of fluid classes with high accuracy, precision and speed.Fluid samples are loaded into wells of a 384-well polypropylene sourceplate and the source plate mounted to a motorized stage system toprovide for automated sampling from any source well. The Echo 555 systemis calibrated for aqueous solutions, including methanol up to 50% inwater as well as up to 50% acetonitrile. The acoustic transducer of thesystem can also be utilized for auto-characterization of a fluid in areservoir, to measure the height of the fluid meniscus as well as otherproperties (e.g., fluid volume, viscosity, density, surface tension,acoustic impedance, acoustic attenuation, speed of sound in the fluid,etc.) to determine optimum parameters for droplet ejection, includingacoustic power, the acoustic frequency, the toneburst duration, and/orthe F-number of the focusing lens.

The fluid reservoirs were wells in 384-well microplates. Added to thefluid reservoirs, i.e., to the sample-loaded wells of the microwellplate, are a suitable biocompatible buffer, e.g., L Tris/EDTA buffer and20 μL BMIM PF₆ as the ionic liquid. Some wells are coated with ahydrophilic, amine coating, and others are not; surfactant is added intosome wells, but not others. An extraction/separation protocol of theinvention is followed, with fluid droplets injected into an invertedmicrowell plate or into an analytical instrument such as a massspectrometer.

1. A method for extracting a target analyte from a fluid sample,comprising: (a) adding the fluid sample to an ionic liquid selected sothat the target analyte preferentially partitions from the fluid sampleinto the ionic liquid and dissolves therein, thereby providing a firstmixture with a first layer comprising a solution of the target analytein the ionic liquid and a second layer deriving from the fluid sample;(b) removing the second layer from the first mixture; (c) combining thesolution of the target analyte in the ionic liquid with an extractionsolvent effective to facilitate preferential partitioning of the targetanalyte from the ionic liquid into the extraction fluid, therebyproviding a second mixture with a third layer comprising the ionicliquid and a fourth layer comprising a solution of the target analyte inthe extraction fluid; and (d) directing a first focused acoustic energytoward the second mixture so as to eject one or more droplets from thethird layer or one or more droplets from the fourth layer.